tert-dodecanethiol

Endpoint summary

Description of key information

A 4 -week oral study in rats, a 4- and 13-week inhalation studies are available using rats, dogs and/or mice were available on t-dodecyl mercaptan.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2018 to 19 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (CD®)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: appr. 10 weeks (males) and appr. 12 weeks (females)
- Weight at study initiation: 396 g to 441 g (males), 257 g to 311 g (females)
- Fasting period before study: No (except for at least 14 hours prior to blood sampling and during urine collection)
- Housing: Animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust.
- Diet: SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 6 days (males), 13 days (females)

DETAILS OF FOOD AND WATER QUALITY: The batches of diet, sawdust and wood shavings were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 December 2018 (males) or 19 December 2018 (females) To: 18 February 2019 (males) or 19 February 2019 (females)

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.
The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight.

Vehicle:

other: 0.5% (w/v) Carboxymethylcellulose (400-800 cps) / 0.5% (v/v) Tween 80 in drinking water treated by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
According to Charles River Laboratories Evreux/Study No. 46439 AHS (homogeneity testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
The test item dose forulations were prepared on the days of treatment, stored at room temperature and protected from light and used within 4 hours after the end of their preparation.

VEHICLE
- Concentration in vehicle: 0, 10, 20 and 40 mg/mL for the control, low, mid and high dose groups, respectively
- Amount of vehicle: 5 mL/kg bw/day
- Batch no. carboxymethylcellulose (400-800 cps): SLBQ8545V
- Batch no. Tween 80: BCBT5201
- Drinking water treated by reverse osmosis using ELIX 5 (Millipore S.A.)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed using an analytical method developed and validated at Charles River Laboratories Evreux (Charles River Laboratories Evreux/Study No. 46438 VAA) prior to dose formulation analysis using High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS).
Dose formulations were analysed three times during the study: Weeks 1, 5 and 9 (referring to the Week 1 of males).

A sample was taken from control and test item dose formulations and analyzed using the validated method.
Acceptance criterion: Measured concentration = nominal concentration ± 15%

Duration of treatment / exposure:

The males were treated starting 4 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed) until euthanasia (11 weeks in total).
The females were treated starting 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), during gestation, during lactation until Day 21 p.p. inclusive until euthanasia for females with no delivery.

Frequency of treatment:

Once daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of the results of a previous study (Charles River Laboratories Evreux/Study No. 46746 TSR) performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg bw/day.
In this study, the test item induced ptyalism at 300 and 1000 mg/kg bw/day, hard/swollen abdomen at 1000 mg/kg bw/day (last day of treatment only), lower body weight gain (-50% vs. controls) and lower food consumption (-35% vs. controls) mainly at 1000 mg/kg bw/day between Days 1 and 4.
At pathology, the test item administration at = 100 mg/kg bw/day induced squamous cell hyperplasia and hyperkeratosis in the forestomach and centrilobular hepatocellular hypertrophy in the liver in both sexes, and tubular hyaline droplets accumulation and degeneration/regeneration in the kidneys in males only. Findings in the forestomach and kidneys were considered to be adverse at = 300 mg/kg bw/day. At 1000 mg/kg bw/day, hepatocellular hypertrophy associated with subcapsular hepatocellular necrosis was considered to be adverse, and adverse ulceration was present in the stomach.
The No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg bw/day and the maximal tolerated dose level to be lower than 300 mg/kg bw/day.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period (including weekends and public holidays)
- Cage side observations included observations for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day
- Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations males: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia;
- Time schedule for examinations females: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8, 13, 17 and 21 p.p. Females euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption of males: measured once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia;
- Food consumption of females: measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8, 8-13, 13-17 and 17-21 p.p.
During the mating period, food consumption was not measured for males or females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included
- Coagulation parameters (Prothrombin time, fibrinogen and activated partial thromboplastin time) were determined from the first five males and lactating females from each group on the day of euthanasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included
- Thyroid hormones (T4 and TSH) were determined for pups sampled on Day 21 p.p. and for F0 males sampled at termination.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (at least 14 hours) before necropsy)
- Metabolism cages used for collection of urine: Yes, urine was collected onto thymol crystals
- Animals fasted: Yes
- All parameters according to guideline were included.

FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations: once at the end of the treatment period (for males: during the last week of treatment; for females: on Day 21 p.p. after euthanasia of the pups)
- Dose groups that were examined: The first five males and lactating females from each group
- Battery of functions tested:
- Detailed clinical examination (including in the cage "touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia;
- The assessment of reactivity to manipulation and to different stimuli (including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature);
- Motor activity (measured once by automated infra-red sensor equipment over a 60 minute period).

Sacrifice and pathology:

GROSS PATHOLOGY:
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
Males: after the end of the mating period (at least 10 weeks of treatment in total),
Females: on Day 22 p.p. Females which did not deliver were euthanized on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery) by inhalation of carbon dioxide gas followed by cervical dislocation since gestation was suspected.

A complete macroscopic post-mortem examination was performed on all F0 animals including those that were euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 22 p.p. and for one low dose female euthanized on Day 26 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

ORGAN WEIGHTS:
Organ weights of the main organs of the first five euthanized as scheduled males and the first five females euthanized on Day 22 p.p. of each group were determined. A table in which all tissue procedures are summarized is attached below.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (attached below) from the first five euthanized as scheduled males and lactating females of the control and high-dose groups,
- all macroscopic lesions of all groups, including kidneys of the prematurely euthanized low dose group female and the subcutis mass of the prematurely euthanized high dose group female,
- reproductive organs from females that did not conceive, or from pregnant female that did not deliver, to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Based upon the results of the microscopic examination of the high-dose group, the following tissues of the low- and intermediate-dose groups were examined on the first five euthanized as scheduled males and/or lactating females: kidneys (males), liver (males and females), thyroids (males and females), stomach with forestomach (males), forestomach (females), vagina (females).

Other examinations:

Monitoring of estrous cycle
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 a.m.):
- during the 2 last weeks of the pre-treatment period (including for the two supplementary females per group, whose data are not presented in the study report),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day of sacrifice before euthanasia, to allow correlation with reproductive organs histopathology.

Statistics:

Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
The statistical analysis sequence for hematology, blood biochemistry, urinalysis, hormones, motor activity, anogenital distance, nipples/areolae, live birth index, sex-ratio and post-implantation loss parameters and for organ weights (level of significance of 0.05 or 0.01) are attached below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was observed in males and females given 100 or 200 mg/kg bw/day during the premating, pregnancy and lactation periods. It was also noted in one isolated female given 50 mg/kg bw/day during the pregnancy period. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

The other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (area of hair loss, cutaneous lesions, scabs), chromodacryorrhea, chromorhinorrhea, reddish vaginal discharge, short/broken teeth, mass on thorax area and/or reflux at dosing, were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No unscheduled deaths occurred in males at any dose level during the study. No mortality of females occurred in the control group and the group dosed at 100 mg/kg bw/day.
At 50 mg/kg bw/day, one female was prematurely euthanized on Day 23 p.c. due to difficulties to deliver. Signs of poor clinical condition (pallor of eyes and extremities, cold to the touch, round back, piloerection and dyspnea) were noted on the day of sacrifice. At necropsy, there were six alive and two dead fetuses in the uterine horns, enlarged and hemorrhagic placenta, red left adrenal gland, red content in vagina and irregular color in kidneys. This latter finding correlated microscopically with a severe acute degeneration/necrosis of tubules and glomeruli. These changes were considered to be unrelated to test item administration in view of their isolated occurrence and the absence of dose-relationship. They could be secondary to uterine/placental infection and sepsis in spite no bacteria were observed at microscopic examination of the kidney.

At 200 mg/kg bw/day, one female was prematurely euthanized on Day 2 p.p. because of the presence of a mass on the mammary gland. This mass of 3.5 x 3 cm, white and heterogeneous in the subcutis (mammary gland region), was a mammary adenocarcinoma and was considered to be unrelated to the test item treatment.

Due to no delivery, three females were euthanized on Day 26 p.c. without clinical signs prior to death (except for ptyalism in the high dose females):
- one female at 50 mg/kg bw/day. At necropsy, one dead fetus and one placenta were found in the left horn;
- two females at 200 mg/kg bw/day. At necropsy, the females were found to be non pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant effects were observed on mean body weights or mean body weight changes in males at any dose level. The lower mean body weight gain recorded in males given 200 mg/kg bw/day throughout the treatment period (-10% vs. controls) was not attributed to the test item treatment as the difference was slight, due to isolated body weight losses and with no impact on the terminal body weight (-3% vs. controls). No relevant effects were observed on mean body weights or mean body weight changes in females at any dose level. A lower mean body weight gain recorded in females given 200 mg/kg bw/day during the gestation period (16% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female which also consumed less food (body weight gain of +20 g on Days 14-20 vs. +94 g in controls).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on mean food consumption were observed in males at any dose level. The mean food consumption was considered to be unaffected by the test item treatment at any dose level.

The lower mean food consumption recorded in females given 200 mg/kg bw/day during the lactation period (-10% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female (food consumption values between 22 and 44 g/day; without these values the difference vs. controls felt to -3%).

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related effects were noted on the hematology parameters at the end of the treatment period.

The only statistically significant differences between control and test item-treated animals (i.e. lower mean cell hemoglobin concentration in females at 50 mg/kg bw/day and males at 100 mg/kg bw/day, lower relative reticulocyte count in males at 50 mg/kg bw/day, higher neutrophil counts in males at 200 mg/kg bw/day, lower eosinophil counts in males at 100 mg/kg bw/day, higher large unstained cell counts in males at 50 and 100 mg/kg bw/day), were not attributed to the test item treatment as they were of low magnitude and/or isolated and/or not dose-related.

No test item-related effects were noted on the clotting parameters at the end of the treatment period. The only statistically significant difference between control and test item-treated animals (i.e. longer activated partial thromboplastin time in males at 100 mg/kg/day) was not attributed to the test item treatment as it was not dose-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related effects were noted on the blood biochemistry parameters at the end of the treatment period.

The only statistically significant differences between control and test item-treated animals were not attributed to the test item treatment as they were of low magnitude and/or isolated (calcium, glucose, creatinine, protein and albumin levels) or were noted with an opposite trend in males and females (triglyceride levels) or because of the direction and magnitude of the changes (total bilirubin and bile acid levels, alanine aminotransferase activity).

No effects were noted on the mean thyroid hormone levels (T4 and TSH plasma levels) in males at termination.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related effects were noted on the quantitative or qualitative urinary parameters at the end of the treatment period.

The only statistically significant difference between control and test item-treated animals (i.e. lower urinary volume in females given 200 mg/kg bw/day) was not attributed to the test item treatment and it was of low magnitude and isolated.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No behavioral or neurological abnormalities were observed during the tests in any group.
The motor activity (60-minute recording period) was unaffected by the test item treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-related increased absolute and/or relative-to-body liver weights were noted in males treated at = 50 mg/kg bw/day (p<0.01 for both absolute and relative-to-body weights at 200 mg/kg bw/day, p<0.01 or 0.05 in relative weights only at 50 or 100 mg/kg bw/day; statistical significance was not reached for the absolute weights at 50 or 100 mg/kg bw/day). This difference was also noted in females treated at 100 mg/kg bw/day (p<0.05) and did not reach statistical significance at 200 mg/kg bw/day. These differences correlated with hepatocellular hypertrophy at microscopic examination.

Dose-related increased absolute and relative-to-body kidney weights were noted in males at = 100 mg/kg bw/day (p<0.01). These differences correlated with tubular hyaline droplet accumulation and dilatation at microscopic examination.

An overview of these results is included below.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic changes.
The few gross changes were considered to be part of the spontaneous background seen in the rat kept under laboratory conditions. This included the yellow mass in the epididymal adipose tissue from one high-dose male that correlated with microscopic granulomatous inflammation (probably secondary to fat torsion). There was also a unilateral small and tan kidney in one high dose female that correlated with a microscopic malformation (congenital).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An overview of the microscopic findings is included below in tabular form below.
Test item-related microscopic observations included mainly adverse renal changes in males (degeneration/necrosis of tubules, accumulation of hyaline droplets, casts, tubular dilatation and basophilia) and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid gland, hyperkeratosis and squamous cell hyperplasia in forestomach, stomach (atrophy of glands) and vagina (increased mucification).

Kidneys: Minimal degeneration/necrosis of proximal tubules, associated with minimal to marked accumulation of hyaline droplets in proximal tubules, casts (granular and, to a lesser extent, hyaline), tubular dilatation in cortex and medulla and cortical basophilia (increased severity/incidence) were noted in males treated at = 50 mg/kg bw/day when compared to controls.

Liver: Minimal to slight hepatocellular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day. It was mostly in centrilobular areas but also sometimes diffuse.

Thyroid: Minimal follicular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day, in association with heterogeneous colloid in 2/5 high-dose males.

Forestomach: Minimal to slight hyperkeratosis often associated with squamous cell hyperplasia were noted in males and females treated at = 50 mg/kg bw/day.

Stomach: Minimal atrophy of gastric gland was noted in 4/5 males treated at 200 mg/kg bw/day. This affected mostly the parietal cells.
Vagina: Increased incidence and severity of vaginal mucification was noted in females treated at 100 or 200 mg/kg bw/day.

Additional examination of males/females that did not conceive:
The males had no lesions in the reproductive organs.
For one female, the left horn contained one dead fetus and the placenta was partially present. There was a moderate acute granulocytic inflammation in the uterus with edema and bacteria in lumen that were probably secondary to the presence of a dead fetus. Two females had functional estrus cycle (one was in proestrus and the other was in estrus). There were no microscopic abnormalities in their reproductive tract.
The other changes were considered not to be related to the test item administration since they were not dose-related, of low incidence and part of the spontaneous background in the rat kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects were observed on the estrous cycle at any dose level during the treatment period.

Details on results:

There was no test item-related intercurrent death during the course of the study. Both unscheduled deaths were related to spontaneous affections (mammary gland adenocarcinoma and renal necrosis probably secondary to uterine infection).

In kidneys, increased severity and incidence of accumulation of hyaline droplets (consistent with a2µglobulin) were noted in males treated at = 50 mg/kg bw/day and correlated with increased weights. In view of the presence of degeneration/necrosis and of the magnitude of the hyaline droplets accumulation (up to marked), these changes were considered to be adverse at all dose-levels. It is noteworthy that the a2µglobulin droplets are specific of the male rat and are not present in humans.

In the liver, hepatocellular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day, correlated with increased weights. The test item was considered to induce hepatocellular enzymatic induction with secondary increased hepatic clearance of thyroid hormones, and thus hypertrophy of thyroid glands, as described in Histopathology of preclinical toxicity studies (Greaves P., 2012, p. 771) This was considered to be of low magnitude, of the absence of associated deleterious changes as degeneration/necrosis, and since there were no clinical pathology correlates, and thus this was considered to be non-adverse.

Follicular hypertrophy was noted in the thyroid glands from males at 50 mg/kg bw/day and from males and females treated at = 100 mg/kg bw/day. This correlated with the increased weights and was linked to the liver hypertrophy (see above). This was considered to be of low magnitude and in the absence of changes in T4 and TSH plasma levels, was considered to be non-adverse.

In the forestomach, the hyperkeratosis and hyperplasia seen at = 50 mg/kg bw/day were considered to be non adverse in view of the low magnitude of these changes and of the absence of associated deleterious lesions (no associated ulceration or inflammation).

In the stomach, the atrophy seen in 4 males at 200 mg/kg bw/day was considered to be non-adverse in view of the low magnitude of this change and of the absence of associated deleterious lesions (no ulceration).

It was considered that the mucification in the vagina at 100 or 200 mg/kg bw/day was non-adverse in view of the low magnitude of this change (up to moderate) and of the presence of this change in 1/5 controls.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

The test item concentrations in the administered dose formulations analyzed in Weeks 1 (two preparations), 5 and 9 (referring to Week 1 for males) remained within an acceptable range of variations of -8.9% to +1.8% when compared to the nominal values (± 15% of the nominal concentrations), except for the first formulation of group 3 male in Week 1 (-41.6%). No test item was detected in the control dose formulation.

Table: Relevant changes in mean final body weights and organ weights in treated groups (% changes from controls)

Sex	Male	Male	Male	Female	Female	Female
Dose level (mg/kg bw/day)	50	100	200	50	100	200
Final body weight	+2	0	-5	-2	-1	+1
Kidneys, absolute	+9	+24**	+26**	0	+4	-2
Kidneys, relative	+7	+25**	+31**	-2	+5	-2
Liver, absolute	+16	+19	+30**	+5	+18*	+16
Liver, relative	+13*	+20**	+35**	+3	+19*	+16

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Conclusions:

Based on the results of a repeated dose toxicity study, combined with screening study for reproduction and development toxicity, performed according to OECD guideline 422 and GLP principles, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males.

Executive summary:

A repeated dose toxicity study combined with screening study for reporduction and development toxicity, was performed with Tert-dodecanethiol according to OECD guideline 422 and GLP principles.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tert-dodecanethiol, daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg bw/day, under a constant dosage volume of 5 mL/kg/day. A control group of ten male and ten female rats received the vehicle only (0.5% Carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions. Males were treated for an overall period of approximately 10 weeks: 4 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 8 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks), gestation (3 weeks) and lactation (3 weeks). Males were euthanized after completion of the mating period. Dams were euthanized on Day 22 p.p. The actual test item concentrations in the dose formulations were confirmed in Weeks 1, 5 and 9 using a validated LC/MS-MS analytical method.

Animals were checked daily for clinical signs and mortality. Detailed clinical examinations were performed weekly. Functional observation battery and motor activity were performed on the first five males and five lactating females during the last days of treatment.

Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated and on the day of sacrifice, before euthanasia.

The animals were paired for mating after two (females) or four (males) weeks of treatment and the dams were allowed to litter and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible five males and five females per litter. Pups not selected on Day 4 p.p. were sampled, euthanized and discarded without further examination.

Hematology, coagulation, blood biochemistry and urinary investigations were performed on the first five males and five lactating females in each group at scheduled euthanasia. Thyroid hormone levels (TSH and T4) were determined in males at sacrifice. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five euthanized males and lactating females in all groups.

No test item-related deaths occurred in males or females during the study. Ptyalism was observed mainly at 100 and 200 mg/kg bw/day throughout the study. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect. The functional observation battery and motor activity were unaffected by the test item treatment. Body weight and food consumption were not impaired by the test item treatment. The estrous cycle was not impacted by the test item treatment. Mating, fertility and delivery data were unaffected by the test item treatment.

At hematology, coagulation, blood biochemistry and urinary investigations, no changes were noted. Thyroid hormone analysis did not reveal any disturbances in males at sacrifice.

At pathology investigations, test item-related increased liver weights were noted in males and/or females at = 50 mg/kg bw/day, and increased kidney weights in males at = 100 mg/kg bw/day. No test item-related macroscopic post-mortem changes were noted. Test item-related microscopic observations included adverse renal changes in males at = 50 mg/kg bw/day (mainly degeneration/necrosis, accumulation of hyaline droplets, tubular vacuolation), and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid glands, hyperkeratosis and squamous cell hyperplasia in forestomach at = 50 mg/kg bw/day in males and at = 100 mg/kg bw/day in females, atrophy of glands in stomach in males treated at 200 mg/kg bw/day and increased mucification in vagina at = 100 mg/kg bw/day in females.

At the dose level of 50 mg/kg bw/day, treatment-related changes were limited to increased liver weight in males associated in males with minimal hepatocellular and thyroid follicular hypertrophies and minimal to moderate hyaline droplet nephropathy. Treatment-related changes consisted of increased liver and kidney weights in males and females associated with minimal to slight (at 200 mg/kg bw/day only) hepatocellular hypertrophy and minimal thyroid follicular hypertrophy in both sexes and moderate to marked hyaline droplet nephropathy in males.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The OECD 443 study is considered to be reliable with a klimisch score of 1. In this study, the animals were exposed longer than in the OECD 422 study (16-17 weeks instead of 4 weeks) so the NOAEL of the OECD 443 is more relevant for repeated toxicity endpoint and for DNEL derivations.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: mean body weights were 299 and 199 grams for males and females, respectively
- Housing: in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Paper, Michigan, USA) and a woodenblock (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG) from a commercial supplier (SDS Special Diets Services, Whitham, England)
- Water: ad libitum, tap water
- Acclimation period: 12-15 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet is analyzed by the supplier for nutrients and contaminants. The feed in the feeders was replaced with fresh portions once weekly and filled up as needed. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) supplied by N.V. Vitens.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 May 2016 To: 31 August 2016 (males exposure group), 2 September 2016 (females exposure group), 11 October 2016 (males recovery group), 13 October 2016 (females recovery group)

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 3.25 - <= 4.49 µm

Geometric standard deviation (GSD):

1.71

Remarks on MMAD:

Only applicable for group 4 (aerosol exposure)
Mean MMAD : 3.67 +/- 0.32 µm
Mean GSD: 1.71 +/- 0.09

Details on inhalation exposure:

EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in nose-only inhalation chambers (a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) consisting of a cylindrical aluminium column, surrounded by a transparent cylinder. The column had a volume of 46.7 (group 2 and 3) or 56.0 liters (group 4) and consisted of a top assembly with the entrance of the unit, a mixing section, two (groups 2 and 3) or three (group 4) rodent tube sections and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control animals (group 1) were exposed in a polypropylene nose-only inhalation chamber (manufactured by P. Groenendijk Kunststoffen BV) with a volume of 48.2 liters, which was similar in construction to the aluminium chambers described above.
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Animals were rotated weekly with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in the experience of the testing laboratory it does not reduce possible stress (Staal et al., 2012). Several empty ports were used for test atmosphere sampling and measurement of oxygen, carbon dioxide, temperature and relative humidity. The remaining ports were closed.
In the experience of the testing laboratory, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was more than 1 liter/min per rat. The air entering the unit was maintained between 22 ± 3°C and the relative humidity between 30% and 70%.

TEST ATMOSPHERE
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the vapor test atmospheres (groups 2 and 3) a liquid flow of test material, controlled by a motor-driven syringe pump (WPI Type SPLG110, SP200iZ or SP220i, World Precision Instruments, Sarasota FL, USA) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, The Netherlands) stream of humidified compressed air, by directing it through a glass evaporator which was kept at a temperature of 65.0 (±1)°C by circulating heated water. The resulting atmosphere was cooled by leading it through a glass coil condenser, controlled at a temperature slightly below ambient (19.5 ± 1°C) to prevent condensation of the test material in the exposure chamber. The test atmosphere was subsequently led to the top inlet of an exposure unit, directed downward towards the noses of the animals, and exhausted at the bottom of the inhalation chamber.
The aerosol test atmospheres (group 4) were generated by nebulization using an air-driven atomizer, which was placed at the top inlet of the exposure chamber. The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump (WPI Type SPLG110). The atomizer used during the range finding study (a Schlick type 970/S, Coburg, Germany) was electrically heated to reduce the aerodynamic particle size, and operated at an air pressure of 1.1 bar. The atomizer used during the main study – designed specifically to generate small-sized liquid aerosols – was operated at an air pressure of 2.5 bar. The atomizers were supplied with a stream of humidified compressed air, which was controlled using a reducing valve and measured using a mass view meter or a mass stream meter (Bronkhorst Hi Tec). During the main study, a mass flow controlled (Bronkhorst Hi Tec) bypass stream of humidified compressed air was added in the top of the exposure unit. The resulting aerosol was directed downward, led to the noses of the animals, and exhausted at the bottom of the inhalation chamber. The procedure for aerosolization was optimized during extensive trial experiments in order to achieve an aerodynamic particle size as close as possible to the recommended range of 1-3 µm MMAD (mass median aerodynamic diameter). Adaptation of various settings resulted in a significant reduction of the particle size; a further decrease in particle size seemed technically not achievable at these conditions.
The exposure unit for the control animals (group 1) was supplied with a stream of humidified compressed air only, which was measured using a mass stream meter.
The animals were placed in the exposure unit after stabilization of the test atmospheres. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ACTUAL CONCENTRATION
The actual concentration of the test substance in the test atmosphere during exposure was determined using a gas chromatographic analytical method (GC/MSD) after sampling on XAD-2 adsorption tubes. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing controlled amounts of test atmosphere through XAD-2 adsorption tubes using a sample flow of 0.2 L/min. Samples with a mean volume of 11.5, 2.3 and 0.6 L were taken from the test atmosphere of group 2, 3 and 4, respectively. The sample flow, controlled using a critical orifice in a vacuum-driven sampling line, was checked before and after each exposure using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). The XAD-2 tubes were desorbed with hexane and the total content of test substance was subsequently determined by GC/MSD. During the study, concentrations were determined three times daily for the low-, mid- and high-concentration test atmosphere, and one blank control sample was included. In addition, the composition of the test material in the test atmosphere samples, as determined by the ratio of C10/C11/C12 components, was analyzed by selected ion monitoring. The composition was determined in all samples taken during the first 10 exposure days of the study; during the remainder of the study, the composition was determined in samples obtained on two days per week. A total carbon analyzer (Sick Maihak EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands) was used to qualitatively monitor the stability of the test atmospheres. Total carbon analysis was, however, not suitable for quantitative analysis and the results are therefore not reported.

TIME TO ATTAIN CHAMBER EQUILIBRATION (T95)
The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This follows from the formula C = C8 (1 – e-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C8 is the steady state concentration.

NOMINAL CONCENTRATION AND GENERATION EFFICIENCY]
The nominal concentration was calculated from the daily consumption of test material (by weight), the duration of test atmosphere generation, and the daily average air flow. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as a percentage of the nominal concentration).

PARTICLE SIZE MEASUREMENT
Particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each aerosol-containing exposure condition (i.e. group 4). Test atmosphere samples were diluted using an aerosol diluter (model 3302A, TSI Incorporated; dilution ratio of 100:1) and subsequently measured by APS. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated (APS user’s manual, 2010).

TOTAL AIR FLOW, TEMPERATURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The chamber air flow, temperature and relative humidity of the test atmosphere were recorded at least hourly during exposure. Air flow was measured by recording the readings of mass flow controllers (groups 2 and 3) and the bypass flow (group4). The airflow of group 1 of the study and the airflow through the atomizer of group 4 of the study were measured using mass stream meters and recorded on a PC every minute using CAN transmitters (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). Temperature and the relative humidity of the test atmospheres were measured continuously during exposure, and recorded on a PC every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH).
The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured twice for each group, i.e. during the first and the last week of the exposure period at a moment when all – or almost all – animals were present in the exposure chamber. In the last week of the exposure period of the study, it was not possible to measure O2 and CO2 at a moment when all animals were placed simultaneously, because of the staggered necropsy dates and the scheduled days for FOB measurements (during which animals were not exposed). Oxygen and carbon dioxide measurements were performed on 25 August 2016, when 5 females of each main group were not exposed (due to FOB). If measured during exposure of all animals, the O2 concentration could have been slightly lower at the end of the exposure period, and the CO2 concentration could have been slightly higher than the values measured on 25 August 2016. However, given the high ventilation rates and the results of the remaining measurements, it was considered inconceivable that the limits of >19% and <1% CO2 – as described in OECD guideline 413 – were exceeded at any moment during the study.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

5 ppm

Remarks:

vapour (target concentration group 2)

Dose / conc.:

25 ppm

Remarks:

vapour (target concentration group 3)

Dose / conc.:

100 ppm

Remarks:

aerosol (target concentration group 4)

Dose / conc.:

5 ppm (analytical)

Remarks:

± 0.3 ; vapour (group 2)

Dose / conc.:

25.1 ppm (analytical)

Remarks:

± 1.4 ; vapour (group 3)

Dose / conc.:

96 ppm (analytical)

Remarks:

± 8.4 ; aerosol (group 4)

No. of animals per sex per dose:

- Main test groups (group 1, 2, 3 and 4): 10
- Recovery groups (group 1 and 4): 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 6 weeks

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS:
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through
the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, and reactions to treatment were recorded.

BODY WEIGHT:
The body weight of each animal was recorded once before the start of the exposure period: on day -3 or -4 for males, and on day -5 and -6 for females. These weights were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (day 0), and twice a week thereafter. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured from day 0 over successive periods of 7 days.

WATER CONSUMPTION:
In response to an apparent increase in water intake in animals of group 4 noticed upon visual inspection of the drinking bottles, water consumption was measured for a period of 1 week, starting on 11 August 2016. Water intake was determined per cage, by weighing the drinking bottles. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -7 to -5) and towards the end of the exposure period in the animals of the control and high-concentration main groups (on days 73 to 75). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

HAEMATOLOGY:
- Haematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. EDTA was used as anticoagulant, except for PT where citrate was used. The samples were discarded after analysis.
- In each sample the following determinations were carried out: haemoglobin (Hb), packed cell volume (PVC), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time (PT), thrombocyte count (platelet count).
- The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of haematology parameters (Hb, PCV, RBC, reticulocytes, thrombocytes, MCV, MCH and MCHC) was extended to animals of the recovery groups.

CLINICAL CHEMISTRY:
- Clinical chemistry was conducted at the end of the treatment period on all surviving rats of the main groups after overnight fasting, at the same time blood samples for haematology were collected. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18°C) to enable reanalysis if necessary and then discarded.
- The following measurements were made: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

URINALYSIS:
- All surviving animals of the main groups were transferred to metabolism cages on the day before sacrifice (one animal per cage) for overnight urine collection. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. Urine samples were discarded after analysis.
- The following determinations were carried out in individual samples: volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of urinalysis parameters was extended to animals of the recovery groups.

NEUROBEHAVIOURAL EXAMINATION:
Neurobehavioral testing was conducted on all animals of the main groups of the study. Detailed clinical examinations (arena testing) were performed prior to the first exposure and then once weekly up to and including week 12. Functional Observational Battery tests (FOB, including arena testing) and spontaneous Motor Activity Assessment (MAA) were performed in week 13 of the study. Since exposure-related changes were observed in animals of the main groups, MAA was extended to animals of the recovery groups.
- FOB: The FOB used in the testing laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Unlike the daily, general clinical observations (intended to detect all abnormalities, signs of ill health or reactions to treatment), the FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor based video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. In the main groups, motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.

Sacrifice and pathology:

GROSS PATHOLOGY:
Surviving animals of the main groups were sacrificed at the end of the exposure period (day 92, main group males on 30 and 31 August 2016, main group females on 1 and 2 September 2016) in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 6-week recovery period (day 134) on 11 (males) and 13 (females) October 2016. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on one female animal of the high concentration recovery group which was humanely sacrificed, because it was suffering from a wound on its back (unrelated to exposure to the test material).

ORGAN WEIGHTS:
- At scheduled sacrifice, the following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying: adrenals, brain, epididymis, heart, kidneys, liver, lungs (left lobe only, because the right lobes were lavaged), ovaries, pituitary gland, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands (and their fluids), spleen, testes, thymus, thyroid, uterus
- Relative organ weights (g/kg body weight) were calculated from the absolute organ weight and the terminal body weight.

HISTOPATHOLOGY:
- For histopathological examination, samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The left lung lobes (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation.
- Tissues: adrenals, aorta, axillary lymph nodes, brain [a], caecum, colon, epididymides (right only), eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs [b], trachea [c], larynx [d], mammary glands (females), cervical lymph nodes, nasopharyngeal tissue [e] (with teeth), nerve peripheral (sciatic nerve), esophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary gland, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestine (duodenum, ileum and jejunum), spinal cord [f], spleen, sternum with bone marrow, stomach [g], sublingual salivary glands, testes (right only), thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all gross lesions
[a] Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
[b] Lungs: The left lung lobe was examined microscopically at three levels.
[c] Trachea: Three levels were examined microscopically (including a longitudinal section through the carina of the bifurcation).
[d] Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
[e] Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]).
[f] Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
[g] Stomach: Non-glandular and glandular parts were examined microscopically.
- Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. In addition, slides of the kidneys of male animals of the control and high-concentration group were also stained immunohistochemically with a monoclonal antibody against a2u-globulin (HYB 339-01, Statens Serum Institut, Kopenhagen, Denmark; antigen retrieval with Proteinase K for 5 minutes). Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration groups (main groups 2 and 3) and the recovery groups (recovery groups 1 and 4) were not processed. The noses of the animals of main groups 2 and 3 were decalcified and embedded in paraffin concurrently with the noses of the animals of main groups 1 (control) and 4 (high concentration).
- Bone marrow: Bone marrow smears were prepared of all animals of the main and recovery groups for possible future examination (except from one female animal which was humanely sacrificed on day 105).
- Preparation of testes: The right testes were preserved in Bouin’s fixative and stained with PAS haematoxylin. The left testes was placed on dry ice and subsequently stored in a freezer (<-70°C) until analysis.
- Examination: All preserved tissues of all animals of the control and high-concentration main groups and of the animal which was sacrificed in moribund condition were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate concentration groups and animals of the recovery groups were examined microscopically. Since exposure-related changes were observed in the kidneys of male animals of the high concentration main group, histopathological examination of the kidneys (H&E and a2u-globulin staining) was extended to male animals of the intermediate concentration groups and recovery groups.

Other examinations:

BRONCHOALVEOLAR LAVAGE (BAL) AND MEASUREMENTS
- The lungs of all animals were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 mL saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 mL saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations. Samples of bronchoalveolar lavage fluid were discarded after analysis.
- Biochemical determinations in BAL fluid: The volume of the supernatant was determined. Total protein, phospholipids, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT) were determined.
- Cellular determinations in BAL fluid: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grünwald Giemsa. The differential cell counts were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
- Since no exposure-related changes were observed in animals of the main groups, investigation of BAL parameters was not extended to the animals of the recovery groups.

SPERM ANALYSIS
- For sperm analysis, the left cauda epididymis and left testis of all males of main groups were used. Since no exposure-related changes were observed in males of the main groups, sperm analysis was not extended to the animals of the recovery groups.
- Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis. For this purpose the cauda epididymis was dissected, weighed and minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for the selected males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained and two hundred spermatozoa of the smear of the males of the control group (group 1) and the males of the high-concentration group (group 4) were examined for morphology. Since treatment-related changes were not observed in the high-concentration group, examination of sperm morphology was not extended to the intermediate concentration groups (or to the recovery groups).
- Testicular sperm count: At scheduled necropsy, the left testes of the same males as used for epididymal sperm analysis were placed on dry ice and subsequently stored in a freezer (<-70°C) until determination of the number of homogenization-resistant spermatids. The testes to be analyzed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The evaluation of homogenization-resistant spermatids was performed in males of the control group (group 1) and males of the high-concentration group (group 4). Since treatment-related changes were not observed in the high-concentration group, evaluation of homogenization-resistant spermatids was not extended to the intermediate concentration groups (or to the recovery groups).

OESTRUS CYCLE EVALUATION
In all female animals of the main groups of the sub-chronic main study, vaginal smears to evaluate the oestrus cycle length and normality were made daily in the three weeks prior to sacrifice, including the day of sacrifice. Since no exposure-related oestrus changes were observed, the cycle evaluations were not extended to the animals of the recovery groups.

Statistics:

See any other information on materials and methods incl. tables

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of the high-concentration group displayed a decreased breathing rate during exposure (Tables 2.1 and 2.2). This finding was generally observed in all animals, was first noted on 6 June 2016 (day 4-7) and throughout the exposure period thereafter (except on 15 and 29 June, and 11 July 2016, when no abnormalities were seen). Occasionally, additional abnormalities were observed during exposure in all animals of the high-concentration group, including lethargy (observed on 7 and 8 June 2016), laboured breathing (on 17 June 2016) and restlessness (on 10 August 2016). The abnormalities observed during exposure in animals of the high-concentration group were no longer seen after the end of the 6-hour exposure. One female of this group showed additional clinical abnormalities – dyspnoea, irregular breathing, lethargy, hunched posture, blepharospasm and piloerection – after about two months of exposure (days 64-69), but these findings were no longer observed towards the end of the exposure period. No further exposure-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed across the groups, and slightly more frequently in females than in males. These are common findings, possibly caused by movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin. Two animals developed a skin wound which was treated by topical application of Vaseline (PurolTM) on a daily basis from day 75 (one female of the control group) or day 85 (one male of the low-concentration group) until scheduled sacrifice.

Mortality:

no mortality observed

Description (incidence):

A female animal of the high-concentration group was humanely sacrificed on day 105, because it was suffering from a wound on its back (unrelated to the exposure). All other animals survived until scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A concentration-related effect on body weight (Tables 4.1 and 4.2) was observed in male animals, which consistently reached the level of statistical significance at the mid- and high-concentration level. At the mid-concentration level, the relative difference with controls was limited (<10% at the end of treatment) and – as can be seen from body weight gain data – mainly the result of a reduced growth during the beginning of the exposure phase; normal growth was observed after the first 2-3 weeks of exposure. At the high-concentration level, the difference in body weight with controls was more substantial (about 15% after three months of treatment), and body weight gain data revealed a consistently reduced growth throughout the exposure period. This difference was the result of a reduced growth during the 5-day periods of consecutive exposure; catch-up growth was observed during the (exposure-free) weekend, but this was apparently insufficient to compensate for the growth depression on exposure days. Normal growth was observed during the 6-week post-exposure recovery period; the relative body weight difference between males of the high-concentration group and controls decreased from about 12% on day 94 to 8% on day 133. No exposure-related changes in body weight or body weight gain were found in female animals. A few statistically significant differences between exposed groups and controls were considered chance findings (lower body weight of high-concentration females on days 98 and 112-126, increased weight gain in low-concentration females on days 24-28, 52-56, 70-73 and in high-concentration females on days 84-87, and a decreased weight gain in low concentration females on days 59-63, 73-77 and in high-concentration females on days 105-108), because a concentration-response relationship or any consistent trend over time was absent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (Table 5) in male animals of the high-concentration group was statistically significantly lower than controls throughout the exposure period. During the 6-week recovery period, differences in food intake were no longer observed. No exposure-related changes in food consumption were observed in females or in males of the low-and mid-concentration group. The few isolated statistically significant differences between exposed females and unexposed controls did not show a concentration-response relationship and/or a consistent change over time, and were therefore considered to be chance findings (decreased food intake on days 0-7 in females of the high-concentration group, and increased food intake on days 21-28 in females of the mid-concentration group).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption, measured for a 1-week period (starting on day 70-73) in animals of the main study in response to apparent inter-group differences noted upon visual inspection of the drinking bottles, was substantially increased in males and females of the high-concentration group. The relative difference with controls, which was slightly smaller during the (exposure-free) weekend, was about 25-30% on average. Water intake in animals of the low- and mid-concentration groups was comparable to control level.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Table 3.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of haematology parameters (Tables 6.1 and 7) revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased percentage of reticulocytes in males of the mid-and high-concentration groups. In the absence of a clear concentration-response relationship and any changes in the main red blood cell parameters, no toxicological relevance was attached to this incidental finding. Also, an increase – rather than a decrease – in the percentage of immature red blood cells is commonly associated with adverse (e.g. anaemic-type) changes.
- Decreased haemoglobin concentration in females of the low-concentration group, which was considered to be a chance finding (unrelated to the exposure), because a concentration-response relationship was absent.
- Decreased mean corpuscular haemoglobin concentration (MCHC) in females of the low- and high-concentration group. Since a clear concentration-response relationship or any consistent changes in main red blood cell parameters (including the parameters from which MCHC is calculated) were not observed, these minor differences from controls were considered to be chance findings.
- Slightly decreased absolute numbers of neutrophils and monocytes in female of the low concentration group, which were considered to be chance findings, because no changes were observed at the higher exposure levels and the percentage distribution of differential white bloods cells was not affected.

Investigation of haematology parameters in animals of the recovery groups (Table 6.2), performed in response to the statistically significant differences observed in animals of the main groups, did not reveal any exposure-related changes. The slightly lower thrombocyte count in females of the high-concentration recovery group was considered to be a chance finding, since corresponding changes were not observed at the end of the exposure phase.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of clinical chemistry parameters (Table 8.1) revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Plasma concentrations of albumin and total protein were slightly increased in males of the high-concentration (+6 and +7%, respectively), and slightly decreased in females of the high-concentration group (-9% and -5%, respectively) ; the latter animals also showed a slightly decreased albumin/globulin ratio (-13%).
- Plasma levels of calcium (+5%) and potassium (+15%) were increased in males of the high-concentration group when compared to controls.

Since statistically significant differences were observed in animals of the main groups, clinical chemistry parameters were also examined in animals of the recovery groups (Table 8.2). Differences observed between exposed animals and controls at the end of the 6-week recovery period were limited to a slightly increased plasma concentration of calcium (+2%) in male animals of the high-concentration group .

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis (Tables 9.1, 10.1 and 11.1), performed at the end of the exposure period, revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Increased urinary volume (x2.3) and decreased specific gravity (-15%) in females of the high-concentration group. A similar tendency was observed in males of the high-concentration group, but the level of statistical significance was not reached.
- Slightly decreased ketone levels in males of the high-concentration group, probably resulting from urinary dilution as a consequence of the increased production.
- Increased amount of amorphous material in the sediment of females of the high-concentration group.
- Decreased crystals and sperm cells in the sediment of males of the high-concentration group. A decrease in urinary crystals is not considered to be of any toxicological significance. In the absence of any changes in epididymal and testicular sperm parameters, no relevance is attached to the apparent decrease in sperm cells detected in urine.

Urinalysis performed in animals of the recovery groups (Table 9.2, 10.2 and 11.2), in response to the changes observed in main groups, did not reveal any adverse exposure-related changes. No toxicological relevance was attached to an apparent increase in urinary pH in males, and a decrease in red blood cell content in females of the high-concentration recovery group, when compared to concurrent controls.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- FUNCTIONAL OBSERVATIONAL BATTERY:
No treatment-related effects were observed from detailed clinical observations or from functional observations in any of the exposed groups during the 13 week treatment period. Therefore, detailed clinical examinations and FOB tests were not performed in animals of the recovery groups. In several females from the control, low- mid-, and high concentration groups tiptoe walking was observed in different weeks of the study. Tiptoe walking was not considered to be related to treatment because it was not consistently observed in the concerned animals from first occurrence towards the end of the study, the severity did not increase over time and it was also observed in the control group. Tiptoe walking is considered to be related to estrus: it is part of the normal behavioral repertoire of the female rat, particularly during estrus.
During weekly detailed clinical observations, occasional (transient) signs were recorded in some males and females of different groups. These signs included, crustiness around the eyes, noses, ears, cheek, tail or on the skin of other parts of the body, ocular red discharge, sparsely haired skin between eyes and ears, cheeks or other parts of the body, soiled fur, dermal wounds on cheek, tail, eyelid or other parts of the body, swollen and/or red ear, nose, toe or around the eyes, missing/bleeding and/or broken toe nails, straub tail, knob on tail and increased reactivity to being handled. Based on the incidence and on the distribution among the different groups, these findings were not considered treatment-related.
Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure (resulting in slight irritation of the skin). In one male of the control group, in two males of the low-concentration group and in two males of the high-concentration group a hunched body position was observed occasionally. This finding was not considered to be related to treatment because it was observed only occasionally in the animals concerned, it was also observed in the control group and it was not consistently observed from first occurrence towards the end of the treatment period.
- MOTOR ACTIVITY ASSESSMENT:
In females of the high-concentration group, the assessment at the end of the exposure period (week 13) revealed a statistically significant decrease in motor activity (-38%). As this single difference, which was reversible within the post-exposure recovery period, was not supported by behavioral changes in measurements of the same functional domain or by any histopathological changes in the nervous system, it was not considered as evidence of neurotoxicity induced by tert-dodecanethiol. In males, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment when tested up to 13 weeks vof exposure.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the exposure period, organ weight data (Tables 15.1, 15.2, 16.1 and 16.2) showed the following statistically significant differences between animals exposed the test material and controls:
- Increased relative weight of the liver in males (about 9% above controls) and females (about 24% above controls) of the high-concentration group. A statistically significantly increased liver weight was also observed in females of the low-concentration group, but not at the mid-concentration level.
- Increased relative weight of the kidneys in males of the high-concentration group. In females, kidney weight was increased at all exposure concentrations; a concentration-response relationship was, however, not observed, and average kidney weight in the control group was relatively low when compared to recent historical controls.
- Slightly decreased relative weight (-13%) of the spleen in males of the high-concentration group.
- Decreased weight (-35%) of the thymus in females of the high-concentration group.
- The increased relative weights of the brain and testes observed in males of the high-concentration group were ascribed to the lower terminal body weights in this group, and the well-known inverse correlation between body weight and relative weight of these organs (Feron et al., 1973; Oshi et al., 1979).

Absolute organ weight changes observed in male animals at the end of the exposure period consisted of decreased weight of the left lung (the right lung lobes were lavaged), heart, spleen and thymus in animals of the mid and high concentration group. These changes should be interpreted carefully, and were considered to be largely the result of the lower terminal body weight of these animals. When compared to concurrent controls, the absolute weights of the liver and kidneys were increased in all groups of exposed females at the end of the exposure period, but a concentration-response relationship was not observed. In females of the high-concentration group, the absolute weight of the thymus was statistically significantly lower than controls.

At the end of the recovery period, the relative weight of the liver was slightly (+6%), but statistically significantly increased in males of the high-concentration group. In the absence of any corresponding changes at the end of the exposure period and any corroborative changes in other parameters, the slight changes in weight of the heart, spleen (relative weight of both organs was increased in high-concentration females) and prostate (decreased absolute weight in high-concentration males) were considered to be chance findings reflecting normal biological variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, no exposure-related macroscopic changes were observed in the animals of the main and recovery groups (Tables 17.1 and 17.2). The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation (Tables 18.1 and 18.2) revealed an exposure-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys of 10/10 high-concentration males. In one animal the kidneys also showed a few dilated tubules containing eosinophilic granular debris in the corticomedullary area. The characteristics of the findings in the kidneys, along with the observation that they occurred in males only, were highly suggestive for accumulation of alpha 2 microglobulins (alpha 2u). To further specify these changes, the kidneys were immunohistochemically stained with a monoclonal antibody against alpha 2u. The kidneys of all males showed positive staining, which was expected, because alpha 2 microglobulins are normal for male rats. In general, the overall positivity (i.e. intensity of the immuno-staining) of the kidneys was not very convincingly higher in the treated animals when compared to the controls. However, detailed examination of the outer cortex revealed that the structures identified as hyaline droplets in the HE stained slides showed a positive reaction in the immuno-staining. Because of these observations, the kidneys of the low- and mid-concentration males and of males of the recovery groups were also processed for histopathological evaluation. Microscopy of those kidneys revealed alpha 2u-positive hyaline droplet accumulation in 6/10 low-concentration males, in 8/10 mid-concentration males, and in 1/10 high-concentration males of the recovery group.

The incidence of alveolar macrophage accumulation in the lungs was generally slightly higher in high-concentration animals when compared to controls, though the difference did not reach the level of statistical significance. The increased incidence may just be caused by coincidental variation of a common background finding, though it cannot be completely excluded that it was related to the exposure. In the absence of any exposure-related changes in BAL fluid or any other histopathological changes in the lungs or other parts of the respiratory tract that could be related to the exposure, the alveolar macrophage accumulation (if treatment-related at all) was judged to be of no toxicological significance and was considered to represent a non-adverse, physiological response to the exposure to the test material.

The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE
- Analysis of bronchoalveolar lavage (BAL) parameters at the end of the exposure period (Tables 12.1, 12.2 and 12.3) did not reveal any adverse exposure-related changes in animals of the main groups. No toxicological relevance was attached to a statistically significantly lower protein content in BAL fluid of females of the high-concentration group (as an increase is protein levels is commonly associated with adverse changes) or in the decreased volume of BAL fluid observed in males of the high-concentration group. In the absence of a concentration-response relationship, an increased percentage of macrophages in BAL fluid of females of the low- and mid-concentration groups was considered to be a chance finding.
- Since no exposure-related changes were observed at the end of the exposure phase, bronchoalveolar lavage parameters were not examined at the end of the recovery period.

SPERM ANALYSIS
No statistically significant, or biologically relevant changes were observed in epididymal sperm motility, epididymal sperm count and epididymal sperm morphology (Table 14.1, 14.2 and 14.3). Homogenization-resistant sperm count and daily testicular sperm production were comparable between males of the high-concentration group and unexposed controls.

OESTRUS CYCLE EVALUATION
No statistically significant differences were observed between exposed groups and unexposed controls (Table 13). One female of the mid-concentration and two females of the high-concentration group had an acyclic oestrus cycle. The time between two oestrus stages suggested a pseudopregnancy. Because these females were considered acyclic, no cycle length and number of complete cycles were determined. Two other females in the high-concentration group could not be evaluated because too many smears contained not enough cells, which was probably also related to pseudopregnancy. Due to the impaired general condition of the animals, the females of the high-concentration group might be more sensitive to induction of pseudopregnancy by mechanical stimulation of the vaginal epithelium during the sampling procedure.

Dose descriptor:

NOAEC

Effect level:

25.1 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

Critical effects observed:

yes

Lowest effective dose / conc.:

5 ppm (analytical)

System:

urinary

Organ:

kidney

other: alpha 2u globulin-related histopathological changes in the kidneys of male animals

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Under the conditions of the current study, inhalation exposure to the tert-dodecanethiol resulted in alpha 2u globulin-related histopathological changes in the kidneys of male animals at all concentrations tested (5.0, 25.1 and 96.0 ppm). A NOEC for this male rat-specific finding was therefore not established. The No-Observed-Adverse-Effect-Concentration (NOAEC) for all other parameters investigated in this sub-chronic inhalation toxicity study with the test substance in rats was placed at the mid-concentration level of 25.1 ppm, based on a weakened general condition of animals exposed at the high concentration of 96.0 ppm as evidenced by exposure-related clinical abnormalities, reduced growth and food consumption (males only) and a reversible decrease in motor activity (females only).

Executive summary:

In a GLP compliant sub-chronic inhalation study performed according to OECD 413, the toxicity of the tert-dodecanethiol upon repeated exposure by inhalation was investigated in Sprague Dawley rats. Four main groups of ten male and ten female rats each were exposed (nose-only) to target concentrations of 0 (control), 5, 25 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of ten male and ten female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure. Endpoints to assess toxicity included clinical, neurobehavioral and ophthalmoscopic observations, growth, food and water consumption, haematology, clinical chemistry, urinalysis and organ weights. Oestrus cyclicity was evaluated during the last three weeks of the exposure period, and sperm analysis was conducted at sacrifice. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobe together with the full respiratory tract and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14-day range finding study. In the sub-chronic main study, the low- and mid-concentration test atmospheres were generated by evaporation at target concentrations of 5 and 25 ppm, respectively. The high-concentration of 100 ppm was generated by aerosolization. The target concentrations were accurately achieved as demonstrated by the results of chemical analysis (GC/MS) of test atmosphere samples. The overall average actual concentrations (± standard deviation) of the test substanceduring exposure were 5.0 (± 0.3), 25.1 (± 1.4) and 96.0 (± 8.4) ppm for the low-, mid-, and high-concentration groups, respectively.

Exposure-related mortality did not occur during the study. A female animal of the high-concentration group was humanely sacrificed, because it was suffering from a wound on its back. Clinical observations revealed a decreased breathing rate during exposure observed in all animals of the high-concentration group by the end of the first week and throughout the exposure period thereafter, which was occasionally accompanied by additional abnormalities (lethargy, laboured breathing and restlessness). These abnormalities were generally no longer observed after exposure; only one female showed additional signs after about two months of exposure, but the animal recovered within a few days. No exposure-related clinical abnormalities were seen in animals of the low- and mid-concentration groups. Neurobehavioral observations (arena and Functional Observational Battery testing) did not reveal any exposure-related abnormalities. A motor activity decrease in female animals of the high-concentration group at the end of the exposure phase was no longer observed at the end of the 6-week post-exposure recovery period. Ophthalmoscopic examination did not reveal any exposure-related ocular abnormalities. Body weight data showed a reduced growth in male animals of the high-concentration group throughout the exposure period; average body weight was about 15% below controls at the end of treatment. Average body weight of males of the mid-concentration group was also slightly below control level, which was the result of a reduced growth during the beginning of the exposure phase; normal growth was observed after the first 2-3 weeks of exposure. No exposure-related body weight changes were observed in female animals. Food consumption was reduced in male animals of the high-concentration group throughout the exposure period. No treatment-related changes in food consumption were observed in males of the low- and mid-concentration groups or in females. Water consumption, measured for a one-week period in response to apparent intergroup differences noted upon visual inspection of the drinking bottles, was increased in males and females of the high-concentration group. Water intake in animals of the low- and mid-concentration groups was comparable to control level. Analysis of haematology parameters did not reveal any adverse exposure-related changes. Investigation of clinical chemistry parameters showed slight changes in animals of the high- concentration group at the end of the treatment period when compared to concurrent controls. Plasma concentrations of albumin and total protein were increased in males, but decreased in female animals, which also showed a slightly decreased albumin/globulin ratio. Plasma levels of calcium and potassium were increased in male animals only. With the exception of a slightly increased plasma calcium concentration in males, all of these changes were fully reversible within the 6-week recovery period. Urinalysis revealed an increased volume and urinary dilution in animals of the high-concentration group, which was in line with the increased water intake. The amount of amorphous material in the sediment was increased in females of the high-concentration group. Urinalysis conducted at the end of the recovery period revealed no exposure-related changes. Analysis of biochemical and cellular parameters in bronchoalveolar lavage fluid at the end of the exposure period did not reveal any adverse changes in response to the exposure to the test material. Oestrus cycle evaluation during the last three weeks of the exposure period showed cyclic abnormalities in a few females of the high-concentration group, which were considered to be the result of slight exposure-related discomfort. Sperm analysis, performed in all males at the end of the exposure period, did not reveal any treatment-related abnormalities in epididymal or testicular sperm parameters. Organ weight data revealed an increased relative (to body weight) weight of the liver and kidneys in males and females of the high-concentration group. Kidney weights were increased in females of all groups exposed to the test material, which was likely related to a relatively low average value in the control group; a concentration-response relationship was not observed. In addition, the relative weight of the spleen was decreased in males of the high-concentration group, and thymus weight was decreased in females of the high-concentration group. With the exception of male kidneys (see below), the changes in organ weights were not associated with any histopathological lesions. At the end of the recovery period, statistically significant differences in organ weight between exposed animals and concurrent controls were limited to a slightly increased relative liver weight in male animals of the high-concentration group. Macroscopic examination at scheduled termination revealed no exposure-related gross pathology. Microscopic examination revealed exposure-related histopathological changes in kidneys of male animals (10/10 high-concentration males, 8/10 mid-concentration and 6/10 low-concentration males). The changes were characterized by accumulations of hyaline droplets in tubular epithelial cells, and one animal also showed a few dilated tubules containing eosinophilic granular debris. These findings, and the fact that the changes were observed in male animals only, were highly suggestive for accumulation of alpha 2 microglobulins, which was confirmed by immunohistochemical staining. At the end of the recovery period, the change was seen in one male only, indicating almost complete recovery. Alpha 2u nephropathy is considered to be a typically male-rat specific phenomenon, which is not relevant for human risk assessment. Microscopic examination of the other organs and tissues did not reveal any adverse histopathological changes which were attributable to the exposure to the test material. Under the conditions of the current study, inhalation exposure to tert-dodecanethiol resulted in alpha 2u globulin-related histopathological changes in the kidneys of male animals at all concentrations tested (5.0, 25.1 and 96.0 ppm). A NOEC for this male rat-specific finding was therefore not established. The No-Observed-Adverse-Effect-Concentration (NOAEC) for all other parameters investigated in this sub-chronic inhalation toxicity study with the test substance in rats was placed at the mid-concentration level of 25.1 ppm, based on a weakened general condition of animals exposed at the high concentration of 96.0 ppm as evidenced by exposure-related clinical abnormalities, reduced growth and food consumption (males only) and a reversible decrease in motor activity (females only).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

207 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: mean body weights were 299 and 199 grams for males and females, respectively
- Housing: in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Paper, Michigan, USA) and a woodenblock (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG) from a commercial supplier (SDS Special Diets Services, Whitham, England)
- Water: ad libitum, tap water
- Acclimation period: 12-15 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet is analyzed by the supplier for nutrients and contaminants. The feed in the feeders was replaced with fresh portions once weekly and filled up as needed. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) supplied by N.V. Vitens.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 May 2016 To: 31 August 2016 (males exposure group), 2 September 2016 (females exposure group), 11 October 2016 (males recovery group), 13 October 2016 (females recovery group)

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 3.25 - <= 4.49 µm

Geometric standard deviation (GSD):

1.71

Remarks on MMAD:

Only applicable for group 4 (aerosol exposure)
Mean MMAD : 3.67 +/- 0.32 µm
Mean GSD: 1.71 +/- 0.09

Details on inhalation exposure:

EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in nose-only inhalation chambers (a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) consisting of a cylindrical aluminium column, surrounded by a transparent cylinder. The column had a volume of 46.7 (group 2 and 3) or 56.0 liters (group 4) and consisted of a top assembly with the entrance of the unit, a mixing section, two (groups 2 and 3) or three (group 4) rodent tube sections and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control animals (group 1) were exposed in a polypropylene nose-only inhalation chamber (manufactured by P. Groenendijk Kunststoffen BV) with a volume of 48.2 liters, which was similar in construction to the aluminium chambers described above.
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Animals were rotated weekly with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in the experience of the testing laboratory it does not reduce possible stress (Staal et al., 2012). Several empty ports were used for test atmosphere sampling and measurement of oxygen, carbon dioxide, temperature and relative humidity. The remaining ports were closed.
In the experience of the testing laboratory, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was more than 1 liter/min per rat. The air entering the unit was maintained between 22 ± 3°C and the relative humidity between 30% and 70%.

TEST ATMOSPHERE
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the vapor test atmospheres (groups 2 and 3) a liquid flow of test material, controlled by a motor-driven syringe pump (WPI Type SPLG110, SP200iZ or SP220i, World Precision Instruments, Sarasota FL, USA) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, The Netherlands) stream of humidified compressed air, by directing it through a glass evaporator which was kept at a temperature of 65.0 (±1)°C by circulating heated water. The resulting atmosphere was cooled by leading it through a glass coil condenser, controlled at a temperature slightly below ambient (19.5 ± 1°C) to prevent condensation of the test material in the exposure chamber. The test atmosphere was subsequently led to the top inlet of an exposure unit, directed downward towards the noses of the animals, and exhausted at the bottom of the inhalation chamber.
The aerosol test atmospheres (group 4) were generated by nebulization using an air-driven atomizer, which was placed at the top inlet of the exposure chamber. The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump (WPI Type SPLG110). The atomizer used during the range finding study (a Schlick type 970/S, Coburg, Germany) was electrically heated to reduce the aerodynamic particle size, and operated at an air pressure of 1.1 bar. The atomizer used during the main study – designed specifically to generate small-sized liquid aerosols – was operated at an air pressure of 2.5 bar. The atomizers were supplied with a stream of humidified compressed air, which was controlled using a reducing valve and measured using a mass view meter or a mass stream meter (Bronkhorst Hi Tec). During the main study, a mass flow controlled (Bronkhorst Hi Tec) bypass stream of humidified compressed air was added in the top of the exposure unit. The resulting aerosol was directed downward, led to the noses of the animals, and exhausted at the bottom of the inhalation chamber. The procedure for aerosolization was optimized during extensive trial experiments in order to achieve an aerodynamic particle size as close as possible to the recommended range of 1-3 µm MMAD (mass median aerodynamic diameter). Adaptation of various settings resulted in a significant reduction of the particle size; a further decrease in particle size seemed technically not achievable at these conditions.
The exposure unit for the control animals (group 1) was supplied with a stream of humidified compressed air only, which was measured using a mass stream meter.
The animals were placed in the exposure unit after stabilization of the test atmospheres. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ACTUAL CONCENTRATION
The actual concentration of the test substance in the test atmosphere during exposure was determined using a gas chromatographic analytical method (GC/MSD) after sampling on XAD-2 adsorption tubes. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing controlled amounts of test atmosphere through XAD-2 adsorption tubes using a sample flow of 0.2 L/min. Samples with a mean volume of 11.5, 2.3 and 0.6 L were taken from the test atmosphere of group 2, 3 and 4, respectively. The sample flow, controlled using a critical orifice in a vacuum-driven sampling line, was checked before and after each exposure using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). The XAD-2 tubes were desorbed with hexane and the total content of test substance was subsequently determined by GC/MSD. During the study, concentrations were determined three times daily for the low-, mid- and high-concentration test atmosphere, and one blank control sample was included. In addition, the composition of the test material in the test atmosphere samples, as determined by the ratio of C10/C11/C12 components, was analyzed by selected ion monitoring. The composition was determined in all samples taken during the first 10 exposure days of the study; during the remainder of the study, the composition was determined in samples obtained on two days per week. A total carbon analyzer (Sick Maihak EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands) was used to qualitatively monitor the stability of the test atmospheres. Total carbon analysis was, however, not suitable for quantitative analysis and the results are therefore not reported.

TIME TO ATTAIN CHAMBER EQUILIBRATION (T95)
The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This follows from the formula C = C8 (1 – e-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C8 is the steady state concentration.

NOMINAL CONCENTRATION AND GENERATION EFFICIENCY]
The nominal concentration was calculated from the daily consumption of test material (by weight), the duration of test atmosphere generation, and the daily average air flow. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as a percentage of the nominal concentration).

PARTICLE SIZE MEASUREMENT
Particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each aerosol-containing exposure condition (i.e. group 4). Test atmosphere samples were diluted using an aerosol diluter (model 3302A, TSI Incorporated; dilution ratio of 100:1) and subsequently measured by APS. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated (APS user’s manual, 2010).

TOTAL AIR FLOW, TEMPERATURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The chamber air flow, temperature and relative humidity of the test atmosphere were recorded at least hourly during exposure. Air flow was measured by recording the readings of mass flow controllers (groups 2 and 3) and the bypass flow (group4). The airflow of group 1 of the study and the airflow through the atomizer of group 4 of the study were measured using mass stream meters and recorded on a PC every minute using CAN transmitters (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). Temperature and the relative humidity of the test atmospheres were measured continuously during exposure, and recorded on a PC every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH).
The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured twice for each group, i.e. during the first and the last week of the exposure period at a moment when all – or almost all – animals were present in the exposure chamber. In the last week of the exposure period of the study, it was not possible to measure O2 and CO2 at a moment when all animals were placed simultaneously, because of the staggered necropsy dates and the scheduled days for FOB measurements (during which animals were not exposed). Oxygen and carbon dioxide measurements were performed on 25 August 2016, when 5 females of each main group were not exposed (due to FOB). If measured during exposure of all animals, the O2 concentration could have been slightly lower at the end of the exposure period, and the CO2 concentration could have been slightly higher than the values measured on 25 August 2016. However, given the high ventilation rates and the results of the remaining measurements, it was considered inconceivable that the limits of >19% and <1% CO2 – as described in OECD guideline 413 – were exceeded at any moment during the study.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

5 ppm

Remarks:

vapour (target concentration group 2)

Dose / conc.:

25 ppm

Remarks:

vapour (target concentration group 3)

Dose / conc.:

100 ppm

Remarks:

aerosol (target concentration group 4)

Dose / conc.:

5 ppm (analytical)

Remarks:

± 0.3 ; vapour (group 2)

Dose / conc.:

25.1 ppm (analytical)

Remarks:

± 1.4 ; vapour (group 3)

Dose / conc.:

96 ppm (analytical)

Remarks:

± 8.4 ; aerosol (group 4)

No. of animals per sex per dose:

- Main test groups (group 1, 2, 3 and 4): 10
- Recovery groups (group 1 and 4): 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 6 weeks

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS:
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through
the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, and reactions to treatment were recorded.

BODY WEIGHT:
The body weight of each animal was recorded once before the start of the exposure period: on day -3 or -4 for males, and on day -5 and -6 for females. These weights were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (day 0), and twice a week thereafter. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured from day 0 over successive periods of 7 days.

WATER CONSUMPTION:
In response to an apparent increase in water intake in animals of group 4 noticed upon visual inspection of the drinking bottles, water consumption was measured for a period of 1 week, starting on 11 August 2016. Water intake was determined per cage, by weighing the drinking bottles. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -7 to -5) and towards the end of the exposure period in the animals of the control and high-concentration main groups (on days 73 to 75). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

HAEMATOLOGY:
- Haematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. EDTA was used as anticoagulant, except for PT where citrate was used. The samples were discarded after analysis.
- In each sample the following determinations were carried out: haemoglobin (Hb), packed cell volume (PVC), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time (PT), thrombocyte count (platelet count).
- The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of haematology parameters (Hb, PCV, RBC, reticulocytes, thrombocytes, MCV, MCH and MCHC) was extended to animals of the recovery groups.

CLINICAL CHEMISTRY:
- Clinical chemistry was conducted at the end of the treatment period on all surviving rats of the main groups after overnight fasting, at the same time blood samples for haematology were collected. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18°C) to enable reanalysis if necessary and then discarded.
- The following measurements were made: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

URINALYSIS:
- All surviving animals of the main groups were transferred to metabolism cages on the day before sacrifice (one animal per cage) for overnight urine collection. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. Urine samples were discarded after analysis.
- The following determinations were carried out in individual samples: volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).
- Since (possible) exposure-related changes were observed in animals of the main groups, investigation of urinalysis parameters was extended to animals of the recovery groups.

NEUROBEHAVIOURAL EXAMINATION:
Neurobehavioral testing was conducted on all animals of the main groups of the study. Detailed clinical examinations (arena testing) were performed prior to the first exposure and then once weekly up to and including week 12. Functional Observational Battery tests (FOB, including arena testing) and spontaneous Motor Activity Assessment (MAA) were performed in week 13 of the study. Since exposure-related changes were observed in animals of the main groups, MAA was extended to animals of the recovery groups.
- FOB: The FOB used in the testing laboratory is adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Unlike the daily, general clinical observations (intended to detect all abnormalities, signs of ill health or reactions to treatment), the FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat. First, measurements were carried out in the cage. The rat’s posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling were rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomoted was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behavior was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consisted of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet were painted lightly and landing foot splay was measured.
- Motor activity: Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor based video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. To this end, each session was divided into 5 time blocks of 6 minutes each. Motor activity tests were recorded on DVD, in order to enable re-analysis of motor activity tests should that be necessary for technical reasons. However, re-analysis was not necessary. Therefore, recordings will be removed after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groups were evenly distributed for motor activity test cage and for time as much as possible. In the main groups, motor activity testing of a squad was conducted immediately after functional observations for that squad had finished.

Sacrifice and pathology:

GROSS PATHOLOGY:
Surviving animals of the main groups were sacrificed at the end of the exposure period (day 92, main group males on 30 and 31 August 2016, main group females on 1 and 2 September 2016) in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 6-week recovery period (day 134) on 11 (males) and 13 (females) October 2016. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on one female animal of the high concentration recovery group which was humanely sacrificed, because it was suffering from a wound on its back (unrelated to exposure to the test material).

ORGAN WEIGHTS:
- At scheduled sacrifice, the following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying: adrenals, brain, epididymis, heart, kidneys, liver, lungs (left lobe only, because the right lobes were lavaged), ovaries, pituitary gland, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands (and their fluids), spleen, testes, thymus, thyroid, uterus
- Relative organ weights (g/kg body weight) were calculated from the absolute organ weight and the terminal body weight.

HISTOPATHOLOGY:
- For histopathological examination, samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The left lung lobes (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation.
- Tissues: adrenals, aorta, axillary lymph nodes, brain [a], caecum, colon, epididymides (right only), eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs [b], trachea [c], larynx [d], mammary glands (females), cervical lymph nodes, nasopharyngeal tissue [e] (with teeth), nerve peripheral (sciatic nerve), esophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary gland, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestine (duodenum, ileum and jejunum), spinal cord [f], spleen, sternum with bone marrow, stomach [g], sublingual salivary glands, testes (right only), thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all gross lesions
[a] Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
[b] Lungs: The left lung lobe was examined microscopically at three levels.
[c] Trachea: Three levels were examined microscopically (including a longitudinal section through the carina of the bifurcation).
[d] Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
[e] Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]).
[f] Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
[g] Stomach: Non-glandular and glandular parts were examined microscopically.
- Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. In addition, slides of the kidneys of male animals of the control and high-concentration group were also stained immunohistochemically with a monoclonal antibody against a2u-globulin (HYB 339-01, Statens Serum Institut, Kopenhagen, Denmark; antigen retrieval with Proteinase K for 5 minutes). Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration groups (main groups 2 and 3) and the recovery groups (recovery groups 1 and 4) were not processed. The noses of the animals of main groups 2 and 3 were decalcified and embedded in paraffin concurrently with the noses of the animals of main groups 1 (control) and 4 (high concentration).
- Bone marrow: Bone marrow smears were prepared of all animals of the main and recovery groups for possible future examination (except from one female animal which was humanely sacrificed on day 105).
- Preparation of testes: The right testes were preserved in Bouin’s fixative and stained with PAS haematoxylin. The left testes was placed on dry ice and subsequently stored in a freezer (<-70°C) until analysis.
- Examination: All preserved tissues of all animals of the control and high-concentration main groups and of the animal which was sacrificed in moribund condition were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate concentration groups and animals of the recovery groups were examined microscopically. Since exposure-related changes were observed in the kidneys of male animals of the high concentration main group, histopathological examination of the kidneys (H&E and a2u-globulin staining) was extended to male animals of the intermediate concentration groups and recovery groups.

Other examinations:

BRONCHOALVEOLAR LAVAGE (BAL) AND MEASUREMENTS
- The lungs of all animals were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 mL saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 mL saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations. Samples of bronchoalveolar lavage fluid were discarded after analysis.
- Biochemical determinations in BAL fluid: The volume of the supernatant was determined. Total protein, phospholipids, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT) were determined.
- Cellular determinations in BAL fluid: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grünwald Giemsa. The differential cell counts were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
- Since no exposure-related changes were observed in animals of the main groups, investigation of BAL parameters was not extended to the animals of the recovery groups.

SPERM ANALYSIS
- For sperm analysis, the left cauda epididymis and left testis of all males of main groups were used. Since no exposure-related changes were observed in males of the main groups, sperm analysis was not extended to the animals of the recovery groups.
- Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis. For this purpose the cauda epididymis was dissected, weighed and minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for the selected males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained and two hundred spermatozoa of the smear of the males of the control group (group 1) and the males of the high-concentration group (group 4) were examined for morphology. Since treatment-related changes were not observed in the high-concentration group, examination of sperm morphology was not extended to the intermediate concentration groups (or to the recovery groups).
- Testicular sperm count: At scheduled necropsy, the left testes of the same males as used for epididymal sperm analysis were placed on dry ice and subsequently stored in a freezer (<-70°C) until determination of the number of homogenization-resistant spermatids. The testes to be analyzed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The evaluation of homogenization-resistant spermatids was performed in males of the control group (group 1) and males of the high-concentration group (group 4). Since treatment-related changes were not observed in the high-concentration group, evaluation of homogenization-resistant spermatids was not extended to the intermediate concentration groups (or to the recovery groups).

OESTRUS CYCLE EVALUATION
In all female animals of the main groups of the sub-chronic main study, vaginal smears to evaluate the oestrus cycle length and normality were made daily in the three weeks prior to sacrifice, including the day of sacrifice. Since no exposure-related oestrus changes were observed, the cycle evaluations were not extended to the animals of the recovery groups.

Statistics:

See any other information on materials and methods incl. tables

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of the high-concentration group displayed a decreased breathing rate during exposure (Tables 2.1 and 2.2). This finding was generally observed in all animals, was first noted on 6 June 2016 (day 4-7) and throughout the exposure period thereafter (except on 15 and 29 June, and 11 July 2016, when no abnormalities were seen). Occasionally, additional abnormalities were observed during exposure in all animals of the high-concentration group, including lethargy (observed on 7 and 8 June 2016), laboured breathing (on 17 June 2016) and restlessness (on 10 August 2016). The abnormalities observed during exposure in animals of the high-concentration group were no longer seen after the end of the 6-hour exposure. One female of this group showed additional clinical abnormalities – dyspnoea, irregular breathing, lethargy, hunched posture, blepharospasm and piloerection – after about two months of exposure (days 64-69), but these findings were no longer observed towards the end of the exposure period. No further exposure-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed across the groups, and slightly more frequently in females than in males. These are common findings, possibly caused by movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin. Two animals developed a skin wound which was treated by topical application of Vaseline (PurolTM) on a daily basis from day 75 (one female of the control group) or day 85 (one male of the low-concentration group) until scheduled sacrifice.

Mortality:

no mortality observed

Description (incidence):

A female animal of the high-concentration group was humanely sacrificed on day 105, because it was suffering from a wound on its back (unrelated to the exposure). All other animals survived until scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A concentration-related effect on body weight (Tables 4.1 and 4.2) was observed in male animals, which consistently reached the level of statistical significance at the mid- and high-concentration level. At the mid-concentration level, the relative difference with controls was limited (<10% at the end of treatment) and – as can be seen from body weight gain data – mainly the result of a reduced growth during the beginning of the exposure phase; normal growth was observed after the first 2-3 weeks of exposure. At the high-concentration level, the difference in body weight with controls was more substantial (about 15% after three months of treatment), and body weight gain data revealed a consistently reduced growth throughout the exposure period. This difference was the result of a reduced growth during the 5-day periods of consecutive exposure; catch-up growth was observed during the (exposure-free) weekend, but this was apparently insufficient to compensate for the growth depression on exposure days. Normal growth was observed during the 6-week post-exposure recovery period; the relative body weight difference between males of the high-concentration group and controls decreased from about 12% on day 94 to 8% on day 133. No exposure-related changes in body weight or body weight gain were found in female animals. A few statistically significant differences between exposed groups and controls were considered chance findings (lower body weight of high-concentration females on days 98 and 112-126, increased weight gain in low-concentration females on days 24-28, 52-56, 70-73 and in high-concentration females on days 84-87, and a decreased weight gain in low concentration females on days 59-63, 73-77 and in high-concentration females on days 105-108), because a concentration-response relationship or any consistent trend over time was absent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (Table 5) in male animals of the high-concentration group was statistically significantly lower than controls throughout the exposure period. During the 6-week recovery period, differences in food intake were no longer observed. No exposure-related changes in food consumption were observed in females or in males of the low-and mid-concentration group. The few isolated statistically significant differences between exposed females and unexposed controls did not show a concentration-response relationship and/or a consistent change over time, and were therefore considered to be chance findings (decreased food intake on days 0-7 in females of the high-concentration group, and increased food intake on days 21-28 in females of the mid-concentration group).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption, measured for a 1-week period (starting on day 70-73) in animals of the main study in response to apparent inter-group differences noted upon visual inspection of the drinking bottles, was substantially increased in males and females of the high-concentration group. The relative difference with controls, which was slightly smaller during the (exposure-free) weekend, was about 25-30% on average. Water intake in animals of the low- and mid-concentration groups was comparable to control level.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Table 3.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of haematology parameters (Tables 6.1 and 7) revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased percentage of reticulocytes in males of the mid-and high-concentration groups. In the absence of a clear concentration-response relationship and any changes in the main red blood cell parameters, no toxicological relevance was attached to this incidental finding. Also, an increase – rather than a decrease – in the percentage of immature red blood cells is commonly associated with adverse (e.g. anaemic-type) changes.
- Decreased haemoglobin concentration in females of the low-concentration group, which was considered to be a chance finding (unrelated to the exposure), because a concentration-response relationship was absent.
- Decreased mean corpuscular haemoglobin concentration (MCHC) in females of the low- and high-concentration group. Since a clear concentration-response relationship or any consistent changes in main red blood cell parameters (including the parameters from which MCHC is calculated) were not observed, these minor differences from controls were considered to be chance findings.
- Slightly decreased absolute numbers of neutrophils and monocytes in female of the low concentration group, which were considered to be chance findings, because no changes were observed at the higher exposure levels and the percentage distribution of differential white bloods cells was not affected.

Investigation of haematology parameters in animals of the recovery groups (Table 6.2), performed in response to the statistically significant differences observed in animals of the main groups, did not reveal any exposure-related changes. The slightly lower thrombocyte count in females of the high-concentration recovery group was considered to be a chance finding, since corresponding changes were not observed at the end of the exposure phase.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of clinical chemistry parameters (Table 8.1) revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Plasma concentrations of albumin and total protein were slightly increased in males of the high-concentration (+6 and +7%, respectively), and slightly decreased in females of the high-concentration group (-9% and -5%, respectively) ; the latter animals also showed a slightly decreased albumin/globulin ratio (-13%).
- Plasma levels of calcium (+5%) and potassium (+15%) were increased in males of the high-concentration group when compared to controls.

Since statistically significant differences were observed in animals of the main groups, clinical chemistry parameters were also examined in animals of the recovery groups (Table 8.2). Differences observed between exposed animals and controls at the end of the 6-week recovery period were limited to a slightly increased plasma concentration of calcium (+2%) in male animals of the high-concentration group .

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis (Tables 9.1, 10.1 and 11.1), performed at the end of the exposure period, revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Increased urinary volume (x2.3) and decreased specific gravity (-15%) in females of the high-concentration group. A similar tendency was observed in males of the high-concentration group, but the level of statistical significance was not reached.
- Slightly decreased ketone levels in males of the high-concentration group, probably resulting from urinary dilution as a consequence of the increased production.
- Increased amount of amorphous material in the sediment of females of the high-concentration group.
- Decreased crystals and sperm cells in the sediment of males of the high-concentration group. A decrease in urinary crystals is not considered to be of any toxicological significance. In the absence of any changes in epididymal and testicular sperm parameters, no relevance is attached to the apparent decrease in sperm cells detected in urine.

Urinalysis performed in animals of the recovery groups (Table 9.2, 10.2 and 11.2), in response to the changes observed in main groups, did not reveal any adverse exposure-related changes. No toxicological relevance was attached to an apparent increase in urinary pH in males, and a decrease in red blood cell content in females of the high-concentration recovery group, when compared to concurrent controls.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- FUNCTIONAL OBSERVATIONAL BATTERY:
No treatment-related effects were observed from detailed clinical observations or from functional observations in any of the exposed groups during the 13 week treatment period. Therefore, detailed clinical examinations and FOB tests were not performed in animals of the recovery groups. In several females from the control, low- mid-, and high concentration groups tiptoe walking was observed in different weeks of the study. Tiptoe walking was not considered to be related to treatment because it was not consistently observed in the concerned animals from first occurrence towards the end of the study, the severity did not increase over time and it was also observed in the control group. Tiptoe walking is considered to be related to estrus: it is part of the normal behavioral repertoire of the female rat, particularly during estrus.
During weekly detailed clinical observations, occasional (transient) signs were recorded in some males and females of different groups. These signs included, crustiness around the eyes, noses, ears, cheek, tail or on the skin of other parts of the body, ocular red discharge, sparsely haired skin between eyes and ears, cheeks or other parts of the body, soiled fur, dermal wounds on cheek, tail, eyelid or other parts of the body, swollen and/or red ear, nose, toe or around the eyes, missing/bleeding and/or broken toe nails, straub tail, knob on tail and increased reactivity to being handled. Based on the incidence and on the distribution among the different groups, these findings were not considered treatment-related.
Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure (resulting in slight irritation of the skin). In one male of the control group, in two males of the low-concentration group and in two males of the high-concentration group a hunched body position was observed occasionally. This finding was not considered to be related to treatment because it was observed only occasionally in the animals concerned, it was also observed in the control group and it was not consistently observed from first occurrence towards the end of the treatment period.
- MOTOR ACTIVITY ASSESSMENT:
In females of the high-concentration group, the assessment at the end of the exposure period (week 13) revealed a statistically significant decrease in motor activity (-38%). As this single difference, which was reversible within the post-exposure recovery period, was not supported by behavioral changes in measurements of the same functional domain or by any histopathological changes in the nervous system, it was not considered as evidence of neurotoxicity induced by tert-dodecanethiol. In males, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment when tested up to 13 weeks vof exposure.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the exposure period, organ weight data (Tables 15.1, 15.2, 16.1 and 16.2) showed the following statistically significant differences between animals exposed the test material and controls:
- Increased relative weight of the liver in males (about 9% above controls) and females (about 24% above controls) of the high-concentration group. A statistically significantly increased liver weight was also observed in females of the low-concentration group, but not at the mid-concentration level.
- Increased relative weight of the kidneys in males of the high-concentration group. In females, kidney weight was increased at all exposure concentrations; a concentration-response relationship was, however, not observed, and average kidney weight in the control group was relatively low when compared to recent historical controls.
- Slightly decreased relative weight (-13%) of the spleen in males of the high-concentration group.
- Decreased weight (-35%) of the thymus in females of the high-concentration group.
- The increased relative weights of the brain and testes observed in males of the high-concentration group were ascribed to the lower terminal body weights in this group, and the well-known inverse correlation between body weight and relative weight of these organs (Feron et al., 1973; Oshi et al., 1979).

Absolute organ weight changes observed in male animals at the end of the exposure period consisted of decreased weight of the left lung (the right lung lobes were lavaged), heart, spleen and thymus in animals of the mid and high concentration group. These changes should be interpreted carefully, and were considered to be largely the result of the lower terminal body weight of these animals. When compared to concurrent controls, the absolute weights of the liver and kidneys were increased in all groups of exposed females at the end of the exposure period, but a concentration-response relationship was not observed. In females of the high-concentration group, the absolute weight of the thymus was statistically significantly lower than controls.

At the end of the recovery period, the relative weight of the liver was slightly (+6%), but statistically significantly increased in males of the high-concentration group. In the absence of any corresponding changes at the end of the exposure period and any corroborative changes in other parameters, the slight changes in weight of the heart, spleen (relative weight of both organs was increased in high-concentration females) and prostate (decreased absolute weight in high-concentration males) were considered to be chance findings reflecting normal biological variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, no exposure-related macroscopic changes were observed in the animals of the main and recovery groups (Tables 17.1 and 17.2). The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation (Tables 18.1 and 18.2) revealed an exposure-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys of 10/10 high-concentration males. In one animal the kidneys also showed a few dilated tubules containing eosinophilic granular debris in the corticomedullary area. The characteristics of the findings in the kidneys, along with the observation that they occurred in males only, were highly suggestive for accumulation of alpha 2 microglobulins (alpha 2u). To further specify these changes, the kidneys were immunohistochemically stained with a monoclonal antibody against alpha 2u. The kidneys of all males showed positive staining, which was expected, because alpha 2 microglobulins are normal for male rats. In general, the overall positivity (i.e. intensity of the immuno-staining) of the kidneys was not very convincingly higher in the treated animals when compared to the controls. However, detailed examination of the outer cortex revealed that the structures identified as hyaline droplets in the HE stained slides showed a positive reaction in the immuno-staining. Because of these observations, the kidneys of the low- and mid-concentration males and of males of the recovery groups were also processed for histopathological evaluation. Microscopy of those kidneys revealed alpha 2u-positive hyaline droplet accumulation in 6/10 low-concentration males, in 8/10 mid-concentration males, and in 1/10 high-concentration males of the recovery group.

The incidence of alveolar macrophage accumulation in the lungs was generally slightly higher in high-concentration animals when compared to controls, though the difference did not reach the level of statistical significance. The increased incidence may just be caused by coincidental variation of a common background finding, though it cannot be completely excluded that it was related to the exposure. In the absence of any exposure-related changes in BAL fluid or any other histopathological changes in the lungs or other parts of the respiratory tract that could be related to the exposure, the alveolar macrophage accumulation (if treatment-related at all) was judged to be of no toxicological significance and was considered to represent a non-adverse, physiological response to the exposure to the test material.

The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE
- Analysis of bronchoalveolar lavage (BAL) parameters at the end of the exposure period (Tables 12.1, 12.2 and 12.3) did not reveal any adverse exposure-related changes in animals of the main groups. No toxicological relevance was attached to a statistically significantly lower protein content in BAL fluid of females of the high-concentration group (as an increase is protein levels is commonly associated with adverse changes) or in the decreased volume of BAL fluid observed in males of the high-concentration group. In the absence of a concentration-response relationship, an increased percentage of macrophages in BAL fluid of females of the low- and mid-concentration groups was considered to be a chance finding.
- Since no exposure-related changes were observed at the end of the exposure phase, bronchoalveolar lavage parameters were not examined at the end of the recovery period.

SPERM ANALYSIS
No statistically significant, or biologically relevant changes were observed in epididymal sperm motility, epididymal sperm count and epididymal sperm morphology (Table 14.1, 14.2 and 14.3). Homogenization-resistant sperm count and daily testicular sperm production were comparable between males of the high-concentration group and unexposed controls.

OESTRUS CYCLE EVALUATION
No statistically significant differences were observed between exposed groups and unexposed controls (Table 13). One female of the mid-concentration and two females of the high-concentration group had an acyclic oestrus cycle. The time between two oestrus stages suggested a pseudopregnancy. Because these females were considered acyclic, no cycle length and number of complete cycles were determined. Two other females in the high-concentration group could not be evaluated because too many smears contained not enough cells, which was probably also related to pseudopregnancy. Due to the impaired general condition of the animals, the females of the high-concentration group might be more sensitive to induction of pseudopregnancy by mechanical stimulation of the vaginal epithelium during the sampling procedure.

Dose descriptor:

NOAEC

Effect level:

25.1 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

Critical effects observed:

yes

Lowest effective dose / conc.:

5 ppm (analytical)

System:

urinary

Organ:

kidney

other: alpha 2u globulin-related histopathological changes in the kidneys of male animals

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

Under the conditions of the current study, inhalation exposure to the tert-dodecanethiol resulted in alpha 2u globulin-related histopathological changes in the kidneys of male animals at all concentrations tested (5.0, 25.1 and 96.0 ppm). A NOEC for this male rat-specific finding was therefore not established. The No-Observed-Adverse-Effect-Concentration (NOAEC) for all other parameters investigated in this sub-chronic inhalation toxicity study with the test substance in rats was placed at the mid-concentration level of 25.1 ppm, based on a weakened general condition of animals exposed at the high concentration of 96.0 ppm as evidenced by exposure-related clinical abnormalities, reduced growth and food consumption (males only) and a reversible decrease in motor activity (females only).

Executive summary:

In a GLP compliant sub-chronic inhalation study performed according to OECD 413, the toxicity of the tert-dodecanethiol upon repeated exposure by inhalation was investigated in Sprague Dawley rats. Four main groups of ten male and ten female rats each were exposed (nose-only) to target concentrations of 0 (control), 5, 25 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of ten male and ten female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure. Endpoints to assess toxicity included clinical, neurobehavioral and ophthalmoscopic observations, growth, food and water consumption, haematology, clinical chemistry, urinalysis and organ weights. Oestrus cyclicity was evaluated during the last three weeks of the exposure period, and sperm analysis was conducted at sacrifice. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobe together with the full respiratory tract and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14-day range finding study. In the sub-chronic main study, the low- and mid-concentration test atmospheres were generated by evaporation at target concentrations of 5 and 25 ppm, respectively. The high-concentration of 100 ppm was generated by aerosolization. The target concentrations were accurately achieved as demonstrated by the results of chemical analysis (GC/MS) of test atmosphere samples. The overall average actual concentrations (± standard deviation) of the test substanceduring exposure were 5.0 (± 0.3), 25.1 (± 1.4) and 96.0 (± 8.4) ppm for the low-, mid-, and high-concentration groups, respectively.

Exposure-related mortality did not occur during the study. A female animal of the high-concentration group was humanely sacrificed, because it was suffering from a wound on its back. Clinical observations revealed a decreased breathing rate during exposure observed in all animals of the high-concentration group by the end of the first week and throughout the exposure period thereafter, which was occasionally accompanied by additional abnormalities (lethargy, laboured breathing and restlessness). These abnormalities were generally no longer observed after exposure; only one female showed additional signs after about two months of exposure, but the animal recovered within a few days. No exposure-related clinical abnormalities were seen in animals of the low- and mid-concentration groups. Neurobehavioral observations (arena and Functional Observational Battery testing) did not reveal any exposure-related abnormalities. A motor activity decrease in female animals of the high-concentration group at the end of the exposure phase was no longer observed at the end of the 6-week post-exposure recovery period. Ophthalmoscopic examination did not reveal any exposure-related ocular abnormalities. Body weight data showed a reduced growth in male animals of the high-concentration group throughout the exposure period; average body weight was about 15% below controls at the end of treatment. Average body weight of males of the mid-concentration group was also slightly below control level, which was the result of a reduced growth during the beginning of the exposure phase; normal growth was observed after the first 2-3 weeks of exposure. No exposure-related body weight changes were observed in female animals. Food consumption was reduced in male animals of the high-concentration group throughout the exposure period. No treatment-related changes in food consumption were observed in males of the low- and mid-concentration groups or in females. Water consumption, measured for a one-week period in response to apparent intergroup differences noted upon visual inspection of the drinking bottles, was increased in males and females of the high-concentration group. Water intake in animals of the low- and mid-concentration groups was comparable to control level. Analysis of haematology parameters did not reveal any adverse exposure-related changes. Investigation of clinical chemistry parameters showed slight changes in animals of the high- concentration group at the end of the treatment period when compared to concurrent controls. Plasma concentrations of albumin and total protein were increased in males, but decreased in female animals, which also showed a slightly decreased albumin/globulin ratio. Plasma levels of calcium and potassium were increased in male animals only. With the exception of a slightly increased plasma calcium concentration in males, all of these changes were fully reversible within the 6-week recovery period. Urinalysis revealed an increased volume and urinary dilution in animals of the high-concentration group, which was in line with the increased water intake. The amount of amorphous material in the sediment was increased in females of the high-concentration group. Urinalysis conducted at the end of the recovery period revealed no exposure-related changes. Analysis of biochemical and cellular parameters in bronchoalveolar lavage fluid at the end of the exposure period did not reveal any adverse changes in response to the exposure to the test material. Oestrus cycle evaluation during the last three weeks of the exposure period showed cyclic abnormalities in a few females of the high-concentration group, which were considered to be the result of slight exposure-related discomfort. Sperm analysis, performed in all males at the end of the exposure period, did not reveal any treatment-related abnormalities in epididymal or testicular sperm parameters. Organ weight data revealed an increased relative (to body weight) weight of the liver and kidneys in males and females of the high-concentration group. Kidney weights were increased in females of all groups exposed to the test material, which was likely related to a relatively low average value in the control group; a concentration-response relationship was not observed. In addition, the relative weight of the spleen was decreased in males of the high-concentration group, and thymus weight was decreased in females of the high-concentration group. With the exception of male kidneys (see below), the changes in organ weights were not associated with any histopathological lesions. At the end of the recovery period, statistically significant differences in organ weight between exposed animals and concurrent controls were limited to a slightly increased relative liver weight in male animals of the high-concentration group. Macroscopic examination at scheduled termination revealed no exposure-related gross pathology. Microscopic examination revealed exposure-related histopathological changes in kidneys of male animals (10/10 high-concentration males, 8/10 mid-concentration and 6/10 low-concentration males). The changes were characterized by accumulations of hyaline droplets in tubular epithelial cells, and one animal also showed a few dilated tubules containing eosinophilic granular debris. These findings, and the fact that the changes were observed in male animals only, were highly suggestive for accumulation of alpha 2 microglobulins, which was confirmed by immunohistochemical staining. At the end of the recovery period, the change was seen in one male only, indicating almost complete recovery. Alpha 2u nephropathy is considered to be a typically male-rat specific phenomenon, which is not relevant for human risk assessment. Microscopic examination of the other organs and tissues did not reveal any adverse histopathological changes which were attributable to the exposure to the test material. Under the conditions of the current study, inhalation exposure to tert-dodecanethiol resulted in alpha 2u globulin-related histopathological changes in the kidneys of male animals at all concentrations tested (5.0, 25.1 and 96.0 ppm). A NOEC for this male rat-specific finding was therefore not established. The No-Observed-Adverse-Effect-Concentration (NOAEC) for all other parameters investigated in this sub-chronic inhalation toxicity study with the test substance in rats was placed at the mid-concentration level of 25.1 ppm, based on a weakened general condition of animals exposed at the high concentration of 96.0 ppm as evidenced by exposure-related clinical abnormalities, reduced growth and food consumption (males only) and a reversible decrease in motor activity (females only).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

795 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity study combined with screening study for reproduction and development toxicity (OECD 422, CRL 2020):

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tert-dodecanethiol, daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg bw/day, under a constant dosage volume of 5 mL/kg/day. A control group of ten male and ten female rats received the vehicle only (0.5% Carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions. Males were treated for an overall period of approximately 10 weeks: 4 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 8 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks), gestation (3 weeks) and lactation (3 weeks). Males were euthanized after completion of the mating period. Dams were euthanized on Day 22 p.p. The actual test item concentrations in the dose formulations were confirmed in Weeks 1, 5 and 9 using a validated LC/MS-MS analytical method.

Animals were checked daily for clinical signs and mortality. Detailed clinical examinations were performed weekly. Functional observation battery and motor activity were performed on the first five males and five lactating females during the last days of treatment.

Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated and on the day of sacrifice, before euthanasia.

The animals were paired for mating after two (females) or four (males) weeks of treatment and the dams were allowed to litter and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible five males and five females per litter. Pups not selected on Day 4 p.p. were sampled, euthanized and discarded without further examination.

Hematology, coagulation, blood biochemistry and urinary investigations were performed on the first five males and five lactating females in each group at scheduled euthanasia. Thyroid hormone levels (TSH and T4) were determined in males at sacrifice. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five euthanized males and lactating females in all groups.

No test item-related deaths occurred in males or females during the study. Ptyalism was observed mainly at 100 and 200 mg/kg bw/day throughout the study. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect. The functional observation battery and motor activity were unaffected by the test item treatment. Body weight and food consumption were not impaired by the test item treatment. The estrous cycle was not impacted by the test item treatment. Mating, fertility and delivery data were unaffected by the test item treatment.

At hematology, coagulation, blood biochemistry and urinary investigations, no changes were noted. Thyroid hormone analysis did not reveal any disturbances in males at sacrifice.

At pathology investigations, test item-related increased liver weights were noted in males and/or females at = 50 mg/kg bw/day, and increased kidney weights in males at = 100 mg/kg bw/day. No test item-related macroscopic post-mortem changes were noted. Test item-related microscopic observations included adverse renal changes in males at = 50 mg/kg bw/day (mainly degeneration/necrosis, accumulation of hyaline droplets, tubular vacuolation), and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid glands, hyperkeratosis and squamous cell hyperplasia in forestomach at = 50 mg/kg bw/day in males and at = 100 mg/kg bw/day in females, atrophy of glands in stomach in males treated at 200 mg/kg bw/day and increased mucification in vagina at = 100 mg/kg bw/day in females.

At the dose level of 50 mg/kg bw/day, treatment-related changes were limited to increased liver weight in males associated in males with minimal hepatocellular and thyroid follicular hypertrophies and minimal to moderate hyaline droplet nephropathy. Treatment-related changes consisted of increased liver and kidney weights in males and females associated with minimal to slight (at 200 mg/kg bw/day only) hepatocellular hypertrophy and minimal thyroid follicular hypertrophy in both sexes and moderate to marked hyaline droplet nephropathy in males.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males.

 

oral 14 -day repeated toxicity study (CRL 2019):

The objective of this preliminary study was to evaluate the potential toxicity of the test item, following daily oral administration (gavage) to rats for 2 weeks in order to determine dose selection for a further study to be performed in this species.

Three groups of five male and five female Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, at dose levels of 100, 300 or 1000 mg/kg/day. One control group of five males and five females received the vehicle only (0.5% Carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed before the beginning of the treatment period and then at once a week. Body weight was recorded once pre-test, on the first day of treatment and then at least twice a week. Food consumption was recorded twice a week during the treatment period. At the end of the treatment period, the animals were euthanized and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on kidneys, liver and stomach with forestomach from all animals.

No mortality occurred during the study. Ptyalism was observed with a dose-related incidence at 300 and 1000 mg/kg/day. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect. At 1000 mg/kg, hard abdomen was observed in 4/5 males and 4/5 females from Day 14, sometimes associated with swelling of the abdomen.

Body weight gain was lower in males given 1000 mg/kg/day on Days 1-4 (-50%), while no body weight gain was recorded in females given 1000 mg/kg/day on Days 1-4. These findings were associated with lower food consumption in males and females on Days 1-4 (-35% vs. controls). A tendency towards lower food consumption was noted in females at 300 mg/kg/day (-12% vs. controls).

At pathology investigations, the test item administration at 1000 mg/kg/day induced adverse centrilobular hepatocellular hypertrophy correlated with increased weights in both sexes and macroscopic enlargement in males, and associated with minimal subcapsular hepatocellular necrosis. In the kidneys from males only, the test item administration induced hyaline droplets accumulation in tubular renal cells, correlated with increased weights and tan discoloration at necropsy, and associated with adverse tubular degeneration/regeneration. In the forestomach, adverse squamous cell hyperplasia and hyperkeratosis, indicative of irritant effects of the test item, correlated with white discoloration and yellow deposits at necropsy, were observed in both sexes and were associated with inflammation and adverse ulceration present in the stomach in one male and one female. At 300 mg/kg/day, adverse hyaline droplet accumulation and tubular degeneration/regeneration in the kidneys, correlated with increased kidney weights, was present in males. Adverse squamous cell hyperplasia and hyperkeratosis was present in the forestomach in both sexes. Non-adverse centrilobular hepatocellular hypertrophy correlated with increased liver weights was observed in both sexes. At 100 mg/kg/day, non-adverse microscopic findings were present in the forestomach (squamous cell hyperplasia and hyperkeratosis) and liver (centrilobular hepatocellular hypertrophy) in both sexes and in the kidneys (tubular hyaline droplets accumulation and degeneration/regeneration) in males only. In the kidneys from males, hyaline droplets accumulation, consistent with a2u globulin nephropathy, and seen at all dose levels, is considered to be non-relevant for human.

At 100 mg/kg/day, no in life effects were noted.

At 300 mg/kg/day, the treatment induced ptyalism in males and lower food consumption in females at treatment initiation.

At 1000 mg/kg/day, the treatment led to ptyalism in both sexes, hard/swollen abdomen and impaired the body weight gain and food consumption at treatment initiation.

At all dose levels, the test item induced squamous cell hyperplasia and hyperkeratosis in the forestomach, centrilobular hepatocellular hypertrophy in the liver in both sexes, and tubular hyaline droplets accumulation and degeneration/regeneration in the kidneys in males only. Findings in the forestomach were considered to be adverse at = 300 mg/kg/day in view of their incidence (all animals) and severity (up to moderate). Findings in the kidneys of males were considered to be adverse at = 300 mg/kg/day in view of their severity (up to moderate hyaline droplets and slight tubular degeneration/regeneration). Hyaline droplets accumulation, consistent with a2u globulin nephropathy, and seen in males at all dose levels, is considered to be non-relevant for human.

At 1000 mg/kg/day, hepatocellular hypertrophy was considered adverse in view of the associated subcapsular hepatocellular necrosis, and adverse ulceration was present in the stomach in two individuals.

Under the experimental conditions of the study, the No Observed Adverse Effect Level was set at 100 mg/kg/day based on the non-adverse effects in the liver, forestomach, and the male rat-specific kidney findings at this dose-level. The Maximal Tolerated Dose level for a further OECD TG 422 study was considered lower than 300 mg/kg/day based on adverse findings in forestomach and male kidneys at this dose level.

 

 

Inhalation route : 13-week toxicity study (TNO 2017)

In a GLP compliant sub-chronic inhalation study performed according to OECD 413 (van Triel, 2017), the toxicity of the tert-dodecanethiol upon repeated exposure by inhalation was investigated in Sprague Dawley rats. Four main groups of ten male and ten female rats each were exposed (nose-only) to target concentrations of 0 (control), 5, 25 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of ten male and ten female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure. Endpoints to assess toxicity included clinical, neurobehavioral and ophthalmoscopic observations, growth, food and water consumption, haematology, clinical chemistry, urinalysis and organ weights. Oestrus cyclicity was evaluated during the last three weeks of the exposure period, and sperm analysis was conducted at sacrifice. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobe together with the full respiratory tract and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14-day range finding study. In the sub-chronic main study, the low- and mid-concentration test atmospheres were generated by evaporation at target concentrations of 5 and 25 ppm, respectively. The high-concentration of 100 ppm was generated by aerosolization. The target concentrations were accurately achieved as demonstrated by the results of chemical analysis (GC/MS) of test atmosphere samples. The overall average actual concentrations (± standard deviation) of the test substance during exposure were 5.0 (± 0.3), 25.1 (± 1.4) and 96.0 (± 8.4) ppm for the low-, mid-, and high-concentration groups, respectively.

Exposure-related mortality did not occur during the study. A female animal of the high-concentration group was humanely sacrificed, because it was suffering from a wound on its back. Clinical observations revealed a decreased breathing rate during exposure observed in all animals of the high-concentration group by the end of the first week and throughout the exposure period thereafter, which was occasionally accompanied by additional abnormalities (lethargy, laboured breathing and restlessness). These abnormalities were generally no longer observed after exposure; only one female showed additional signs after about two months of exposure, but the animal recovered within a few days. No exposure-related clinical abnormalities were seen in animals of the low- and mid-concentration groups. Neurobehavioral observations (arena and Functional Observational Battery testing) did not reveal any exposure-related abnormalities. A motor activity decrease in female animals of the high-concentration group at the end of the exposure phase was no longer observed at the end of the 6-week post-exposure recovery period. Ophthalmoscopic examination did not reveal any exposure-related ocular abnormalities. Body weight data showed a reduced growth in male animals of the high-concentration group throughout the exposure period; average body weight was about 15% below controls at the end of treatment. Average body weight of males of the mid-concentration group was also slightly below control level, which was the result of a reduced growth during the beginning of the exposure phase; normal growth was observed after the first 2-3 weeks of exposure. No exposure-related body weight changes were observed in female animals. Food consumption was reduced in male animals of the high-concentration group throughout the exposure period. No treatment-related changes in food consumption were observed in males of the low- and mid-concentration groups or in females. Water consumption, measured for a one-week period in response to apparent intergroup differences noted upon visual inspection of the drinking bottles, was increased in males and females of the high-concentration group. Water intake in animals of the low- and mid-concentration groups was comparable to control level. Analysis of haematology parameters did not reveal any adverse exposure-related changes. Investigation of clinical chemistry parameters showed slight changes in animals of the high- concentration group at the end of the treatment period when compared to concurrent controls. Plasma concentrations of albumin and total protein were increased in males, but decreased in female animals, which also showed a slightly decreased albumin/globulin ratio. Plasma levels of calcium and potassium were increased in male animals only. With the exception of a slightly increased plasma calcium concentration in males, all of these changes were fully reversible within the 6-week recovery period. Urinalysis revealed an increased volume and urinary dilution in animals of the high-concentration group, which was in line with the increased water intake. The amount of amorphous material in the sediment was increased in females of the high-concentration group. Urinalysis conducted at the end of the recovery period revealed no exposure-related changes. Analysis of biochemical and cellular parameters in bronchoalveolar lavage fluid at the end of the exposure period did not reveal any adverse changes in response to the exposure to the test material. Oestrus cycle evaluation during the last three weeks of the exposure period showed cyclic abnormalities in a few females of the high-concentration group, which were considered to be the result of slight exposure-related discomfort. Sperm analysis, performed in all males at the end of the exposure period, did not reveal any treatment-related abnormalities in epididymal or testicular sperm parameters. Organ weight data revealed an increased relative (to body weight) weight of the liver and kidneys in males and females of the high-concentration group. Kidney weights were increased in females of all groups exposed to the test material, which was likely related to a relatively low average value in the control group; a concentration-response relationship was not observed. In addition, the relative weight of the spleen was decreased in males of the high-concentration group, and thymus weight was decreased in females of the high-concentration group. With the exception of male kidneys (see below), the changes in organ weights were not associated with any histopathological lesions. At the end of the recovery period, statistically significant differences in organ weight between exposed animals and concurrent controls were limited to a slightly increased relative liver weight in male animals of the high-concentration group. Macroscopic examination at scheduled termination revealed no exposure-related gross pathology. Microscopic examination revealed exposure-related histopathological changes in kidneys of male animals (10/10 high-concentration males, 8/10 mid-concentration and 6/10 low-concentration males). The changes were characterized by accumulations of hyaline droplets in tubular epithelial cells, and one animal also showed a few dilated tubules containing eosinophilic granular debris. These findings, and the fact that the changes were observed in male animals only, were highly suggestive for accumulation of alpha 2 microglobulins, which was confirmed by immunohistochemical staining. At the end of the recovery period, the change was seen in one male only, indicating almost complete recovery. Alpha 2u nephropathy is considered to be a typically male-rat specific phenomenon, which is not relevant for human risk assessment. Microscopic examination of the other organs and tissues did not reveal any adverse histopathological changes which were attributable to the exposure to the test material. Under the conditions of the current study, inhalation exposure to tert-dodecanethiol resulted in alpha 2u globulin-related histopathological changes in the kidneys of male animals at all concentrations tested (5.0, 25.1 and 96.0 ppm). A NOEC for this male rat-specific finding was therefore not established.The No-Observed-Adverse-Effect-Concentration (NOAEC) for all other parameters investigated in this sub-chronic inhalation toxicity study with the test substance in rats was placed at the mid-concentration level of 25.1 ppm, based on a weakened general condition of animals exposed at the high concentration of 96.0 ppm as evidenced by exposure-related clinical abnormalities, reduced growth and food consumption (males only) and a reversible decrease in motor activity (females only).

 

Inhalation route : 4-week toxicity studies

In a 4-week inhalation toxicity study [similar to OECD TG 407], male and female Sprague-Dawley rats were exposed to a vehicle control or tert-dodecanethiol for 6 hours per day, 5 days per week at nominal concentrations of 26 (0.22 mg/L) and 98 ppm (0.81 mg/L) (IDRC 1985a). The high exposure concentration was a saturated vapour. Male rats showed a statistically significant decrease in body weight gain and a corresponding depression in food consumption at the high exposure concentration. Rats exhibited dark red or black material around their eyes and nose in both exposure groups. Male rats at the high exposure concentration showed a statistically significant increase in creatinine, and liver weights showed an exposure-related increase. Since no macroscopic or microscopic changes in livers were noted, these weight increases were not considered adverse. Male rats at both exposure concentrations exhibited mild renal tubular degeneration and granular cysts which were consistent with species-specific hydrocarbon nephropathy. Female rats at th high exposure concentration exhibited hydronephrosis. Because no biochemical evaluation of the presence of a 2µ globulin was conducted and because kidney effects were seen in females at a higher dose, the renal changes observed in males were considered adverse and potentially applicable to humans.

Therefore, a LOAEC was determined to be 26 ppm (0.21 mg/L) for male rats and a NOAEC was determined to be 26 ppm (0.21 mg/L) for female rats.

 

In a 4-week inhalation toxicity study [similar to OECD TG 407], male and female beagle dogs (3/sex/group) were exposed to tert-dodecanethiol for 6 hours/day, 5 days per week at nominal concentrations of 0 (sham control), 25 (0.21 mg/L) and 106 ppm (0.90 mg/L) (IDRC 1985b). The high exposure concentration was an aerosol. Female dogs in the high exposure concentration showed statistically significant increases in ALT levels and statistically significant reductions in blood urea nitrogen. Both sexes showed statistically significant increases in alkaline phosphatase. There were exposure-related increases in liver weights. Also noted was microscopic hepatocellular hypertrophy at the high exposure concentration for both sexes. Based on liver-related effects observed at 106 ppm, the NOAEC for systemic toxicity was 25 ppm (0.21 mg/L) in dogs.

 

In a 4-week inhalation toxicity study [similar to OECD TG 407], male and female CD-1 mice (10/sex/exposure) were exposed to tert-dodecanethiol for 6 hours/day, 5 days per week at nominal concentrations of 0 (sham control), 25 (0.21 mg/L) and 109 ppm (0.90 mg/L) (IDRC 1985c). The high exposure concentration was a saturated vapour. One male and 1 female mouse died at the high exposure concentration on exposure day 23; however, no cause of death could be determined. Mice in the high exposure concentration group showed signs of alopecia and dried, peeling skin and histopathologic evaluation revealed dermal inflammation, acanthosis, and hyperkeratosis. After 4 weeks of exposure, mice at the high exposure concentration exhibited statistically significant reductions in erythrocyte counts and hematocrit, and statistically significant increases in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) levels. Females exposed to 109 ppm (0.90 mg/L) showed statistically significant increases in ALT and blood urea nitrogen, and decreased alkaline phosphatase values. These females also had statistically significant decreased ovary weights, and histopathological evaluation revealed either an absence of, or few, corpora lutea. Low exposure concentration females showed statistically significant increases in blood glucose. Liver weights revealed exposure-related increases; both sexes showed liver enlargement, discoloration and hepatocellular hypertrophy at the low and high exposure concentrations.Based on liver findings in both sexes and exposure concentrations, the LOAEC for systemic toxicity was 25 ppm (0.21 mg/L) in mice.

 

 

7-days Oral toxicity study in rabbit (CRL 2020)

The objective of this non-GLP preliminary study was to evaluate the potential toxicity of the test item following daily oral administration in the non-pregnant female rabbit, and to establish a Maximum Tolerated Dose (MTD), in order to select dose levels for a further preliminary toxicity study in pregnant rabbits with the same route of administration. Three non-pregnant female New Zealand White rabbits received the test item, at 300 then 1000 mg/kg/day in the vehicle [0.5% Carboxymethylcellulose (400-800 cps) / 0.5% Tween 80 in drinking water treated by reverse osmosis] by oral route (gavage) once daily for 7 days each dose. There was no wash-out period between both doses. A constant dosage volume of 3 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 2 to 3 days. At the end of the treatment period, the females were euthanized and submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

There were no unscheduled deaths during the study. At 300 mg/kg/day, animals did not exhibit any clinical signs but lost weight between Days 1 and 7 (from -5% to -11% when compared to their body weight recorded on the first day of treatment). One female had strongly reduced food consumption since Day 3 and almost no food consumption since Day 5. Minimal to no effect was noticed in the two other animals. At 1000 mg/kg/day, absence of feces was recorded in all the animals for 1 to 3 days at the end of the treatment period. Absence of urine was also noticed in one animal from Day 12.

All the animals lost weight between Days 8 and 15 (from -6% to -12% when compared to their body weight recorded on Day 8). Food consumption was strongly impacted in all three females since the first day of the treatment period (i.e. Day 8). One female did not eat and the two other had gradually decreased food consumption until the end of the study. On Day 14, none of the animals ate (pelleted diet, hay or carrots) and their water consumption was also strongly reduced. At necropsy, all the animals displayed a liver discoloration and a distended colon. These severe effects were considered to be test item-related.

 

In summary, at 300 mg/kg/day, no clinical sign was observed but females lost weight and absence of food consumption was noticed for a few days for one of them. At 1000 mg/kg/day, some clinical signs were noticed, related to a body weight loss and nearly no food or water consumption. At necropsy, all the animals displayed a liver discoloration and a distended colon.

Therefore, the dose level of 1000 mg/kg/day was considered to exceed the Maximum Tolerated Dose (MTD) under the experimental conditions of the study and doses for a further study should not exceed 300 mg/kg/day.

Justification for classification or non-classification

Based on the available data, no classification for repeated toxicity is required for tert-dodecanethiol according to the Regulation EC N°1272/2008.