2-ethylhexyl nitrate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In an OECD 421 Reproduction / Developmental Toxicity Screening Test, the test item, 2-EHN (CAS No. 27247-96-7), was administered daily by oral gavage to male and female Sprague-Dawley rats, from before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 20, 100 or 500 mg/kg/day.

Systemic toxicity was observed at all dose-levels, as evidenced by reduced body weight gain during, at least, the premating period (at least 13% less weight gained than the controls during the first 2 weeks of treatment) (statistically significant at 500 mg/kg/day). As body weight gains which are reduced by more than 10% are considered biologically significant, all groups had biologically significantly reduced body weight gains. Clinical signs of hypoactivity and half-closed eyes were also observed at 100 and 500 mg/kg/day and food consumption was lower than controls’ for females given 500 mg/kg/day.

There were no effects of treatment on pairing but a relationship between development of the fetuses and pups and treatment cannot be excluded because there was one female with only implantation scars and a dead fetus in the uterine horns and another female which delivered a litter of small pups which consequently died on day 3 post-partum.

Mean pup body weight at 500 mg/kg/day was less than that of the controls on day 1 post-partum and pup body weight gain was reduced at 100 and 500 mg/kg/day.

There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day, and the NOAEL for toxic effect on reproductive performance and on progeny was 100 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 May 2005 to 28 June 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines (observed deviation was not considered to have compromised the validity of the study and results), adequate coherence between data, comments and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

food consumption of all male groups was not recorded after mating until sacrifice in error

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3500 (Reproduction/Developmental Toxicity Screening test)

Deviations:

yes

Remarks:

as above

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: (P) x 11 wks
- Weight at study initiation (mean): (P) Males: 414 g; Females: 250 g; (F1) Males: 23 g; Females: 9 g
- Fasting period before study: no
- Housing: suspended wire-mesh cages with a metal tray containing autoclaved sawdust (females were transferred to polycarbonate cages containing autoclaved wood shavings as nesting material on approximately day 14 post-coitum)
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: for a period of 16 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50% +/- 20%
- Air changes (per hr): 12 cylces/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2 June 2005 To: 19 July 2005

Route of administration:

oral: gavage

Vehicle:

other: 0.5% tween 80, prepared using purified water and tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 2, 10 and 50 mg/mL, according to the specific study procedure. The test item was administered as a suspension in the vehicle. The test item dosage formulations were prepared for use up to nine days and stored at +4°C, protected from light, with Teflon gaskets inside the bottle caps.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not indicated
- Concentration in vehicle: 2, 10 and 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurs or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- Mated for up to 14 days
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosage formulations were checked for homogeneity, stability and concentrations.
Check of homogeneity: on dosage forms were prepared at 2, 3 and 5 mg/mL
Check of stability: Each dosage form prepared for homogeneity analysis was sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light)
Check of concentration: in samples taken from each dosage form (including the control) prepared for use in weeks 1, 3 and 6.
Method: validated Gas Liquid Chromatography with Flame Ionisation Detection (GC/FID), Limit of Quantification = 0.02 mg/mL

Duration of treatment / exposure:

In the males:
− 15 days before mating,
− during the mating period (2 weeks),
− until sacrifice (i.e. at least 4 weeks in total),

In the females:
− 15 days before mating,
− during the mating period (2 weeks),
− during pregnancy,
− during lactation until day 5 post-partum inclusive (until sacrifice).

Actual duration: 34 days (males), 42-47 days (females)

Frequency of treatment:

Treatment was administered once daily (7 days per week)

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: [...] weeks: not indicated

Remarks:

Doses / Concentrations:
0, 20, 100, 500 mg/kg/day
Basis:
other: nominal ingested (there was a satisfactory agreement between the nominal and actual concentrations of the test item in the administered dosage forms)

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 in conjunction with the Sponsor on the basis of the results of a 28-day toxicity study by oral route performed in the same species in which animals received 1, 20, 100 and 500 mg/kg/day.
A significant decrease in the body weight of high-dose females was observed in the last week of treatment in addition to a reduced food consumption.Males from the 500 mg/kg/day group exhibited a significant increase in mean platelet number whereas females exhibited a decreased chloride concentration. A number of alterations were observed in the urinalysis of mid- and high-dose groups. There were variations in organ weights, including the liver and kidney, in animals of both sexes, and adrenals in the 500 mg/kg/day females. Microscopic observations were limited to the kidneys of the mid- and high-dose males, including the appearance of hyaline droplets in the proximal tubular epithelium and regenerative changes of the renal tubules.

- Rationale for animal assignment (if not random):
Animals were allocated to groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.

Positive control:

None.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and at least once daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment, then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mating (or until sacrifice) and on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was recorded once a week, over a 7-day period, from the first day of treatment until pairing for mating. The quantity of food consumed by each female was recorded once a week, over a 7-day period, from the first day of treatment through
gestation (days 0-7, 7-10, 10-14, 14-17 and 17-20 post-coitum intervals) and lactation (days 1-5 post-partum interval) until sacrifice. During the mating period, the food consumption was not recorded in either males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

No.

Sperm parameters (parental animals):

Parameters examined in all males of parental generation:
[testis weight, epididymis weight,
in control and high dose groups of male parental generation, the tailed and round spermatids, spermatocytes, spermatogonia and the different stages of the spermatogenic cycle were evaluated. In addition, attention was paid to the testicular interstitial cells.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, number of live, dead and cannibalized pups, presence of gross anomalies, weight gain, clinical signs]

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
[yes, for external and internal abnormalities]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the end of the mating period
- Maternal animals: all surviving animals = day 6 post-partum or, for females which had not delivered by day 25 post-coitum, day 25 or day 26 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on the ovaries, testes and epididymides of all males and females of the control and high-dose groups.
The tailed and round spermatids, spermatocytes, spermatogonia and the different stages of the spermatogenic cycle were evaluated. In addition, attention was paid to the testicular interstitial cells.
The testes and epididymides of all the males were weighed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on day 6 post-partum.
- These animals (and pups found dead) were subjected to postmortem macroscopic examinations.

GROSS NECROPSY
- Performed on all pups (surviving and found dead).

HISTOPATHOLOGY / ORGAN WEIGTHS
There was neither microscopic examination nor organ weighing.

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

hypoactivity, loud breathing, ptyalism, half-closed eyes, piloerection, round back and emaciated appearance

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

biologically significant reduced body weight gain during, at least, the premating period, at all dose-levels

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

biologically significant reduced body weight gain during, at least, the premating period, at all dose-levels

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male given 500 mg/kg/day had hypoactivity and half-closed eyes on day 15 of dosing while another had the same signs on days 12, 13 and 14 of dosing and loud breathing on day 26. All males had ptyalism from day 5 until the end of the treatment period. The deviation of the ptyalism was measured to be 4-5 hours after dosing, and may be related to the taste of the test item.
Two males given 100 mg/kg/day had ptyalism on day 25 of dosing while another had it on days 14, 15 and 16 and again between day 24 and day 27.
There were no clinical signs observed at 20 mg/kg/day.

Premating
Three females given 500 mg/kg/day had hypoactivity and half-closed eyes sporadically towards the end of the second week of dosing. One of these females (H25044) also had piloerection, round back and emaciated appearance on days 15 and 16 of dosing. All females had ptyalism from day 5 of dosing until the end of the premating period.
One female given 100 mg/kg/day had hypoactivity and half-closed eyes on day 12 of dosing. Seven of the ten females at this dose-level had ptyalism towards the end of the second week of dosing and the beginning of the third week.

Gestation
During the gestation period at 500 mg/kg/day, three females had hypoactivity and half-closed eyes at the beginning and (for two females) also at the end of gestation. One of the females also had loud breathing on day 8 post-coitum. Female H25044 again had round back and emaciated
appearance for the first four to six days of gestation. All females had ptyalism throughout the gestation period.
At 100 mg/kg/day, seven females had ptyalism, generally during the first half of the gestation period, but also towards the end for some females.

Lactation
At 500 mg/kg/day, two females had hypoactivity and half-closed eyes for up to 5 days and all females had ptyalism until sacrifice on day 6 post-partum.
At 100 mg/kg/day, one female had ptyalism on day 0 post-partum only. Areas of hairloss were observed, also in the control animals, but as this is commonly seen in rats kept under laboratory conditions, it was considered not to be related to treatment.
There were no clinical signs observed at 20 mg/kg/day during the study.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was a dose-related lowering of body weight gain for males and females at all dose-levels during the 2 week premating period, achieving statistical significance at 500 mg/kg/day. During the gestation period, females given 500 mg/kg/day continued to gain less weight than the controls (p<0.01) resulting in statistically significantly low mean body weights on day 20 post-coitum (p<0.01) and days 1 and 5 post-partum (p<0.05), while the other groups gained comparable amounts. During lactation, all female groups gained approximately comparable amounts of weight to the controls.

Male food consumption at all dose-levels was comparable with the controls throughout the study.
During the first week of dosing, females given 500 mg/kg/day consumed statistically significantly less food than the controls (-19%, p<0.001). During the second week, food consumption was still slightly reduced (-14%, not statistically significant). Food consumption remained less than the controls throughout gestation and lactation, achieving statistical significance from day 7 to day 10 and from day 14 to day 20 post-coitum.
There was no effect of treatment on food consumption at 20 or 100 mg/kg/day.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All pairs mated and the mean number of days taken to mate was comparable with the controls for all groups.
There was one non-pregnant female in each of the groups given 20 or 500 mg/kg/day. The remaining females were pregnant, although one female (H25037) in the 500 mg/kg/day dose group did not deliver and was found to have one dead fetus and nine implantation scars in the uterine horns at necropsy. From the poor body weight performance of this female during the gestation period, an unseen delivery was not suspected and therefore it is considered that the scars were implantations resorbed very early during the pregnancy.
The duration of gestation was similar between the control and test item-treated groups and close to the normal value of 21 days. There was no effect of treatment on the mean number of liveborn pups at any dose-level or on pup death after birth at 20 and 100 mg/kg/day. One female (H25045) in the 500 mg/kg/day dose group had a dead litter on day 3 post-partum and, as a result, the number of pups dying was statistically significantly higher at 500 mg/kg/day than for the control group.


ORGAN WEIGHTS (PARENTAL ANIMALS)
The differences observed were considered to be of no toxicological importance.


GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related necropsy findings were noted. The few necropsy findings encountered were recognized as those commonly recorded changes in the rat of this strain and age in this laboratory and none were considered to be of toxicological importance.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Morphological examination of the testes: no treatment-related abnormalities were found in the test-treated animals.
Ovaries: the follicular development and the number of corpora lutea were similar in both control and treated animals and showed no disturbance by treatment with the test item.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

at 500 mg/kg/day, one pup was found dead on day 1 post-partum, 11 pups were found dead on day 2 post-partum and the one remaining pup was found dead on day 3 post-partum

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

lower body weight gain at all dose-levels

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
From one litter (dose-level of 500 mg/kg/day) one pup was found dead on day 1 post-partum, 11 pups were found dead on day 2 post-partum and the one remaining pup was found dead on day 3 post-partum. No clinical signs had been observed in the pups prior to death.

CLINICAL SIGNS (OFFSPRING)
One pup in the 20 mg/kg/day dose group had no tail. As this was an isolated finding and not also observed in the high-dose group, it was considered to be spontaneous in origin.
All other clinical observations (cold to the touch, anouria, tail necrosed, emaciated appearance, wound on abdomen...) were those commonly seen in pups of this age and as they were often also seen in control pups, they were considered not to be related to treatment.

BODY WEIGHT (OFFSPRING)
Pup weight gain from day 1 to day 5 post-partum was slightly lower than the controls’ at 100 mg/kg/day (-10%) and more markedly lower at 500 mg/kg/day (-22%). Pup body weights were lower than the controls’ on days 1 and 5 post-partum at 500 mg/kg/day and on day 5 post-partum at 100 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING)
There were no treatment-related gross findings.

OTHER FINDINGS (OFFSPRING)
The percentage of male pups was slightly low at 500 mg/kg/day, when compared with the controls.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

Systemic toxicity was observed at all dose-levels, as evidenced by reduced body weight gain during, at least, the premating period (at least 13% less weight gained than the controls during the first 2 weeks of treatment) (statistically significant at 500 mg/kg/day). As body weight gains
which are reduced by more than 10% are considered biologically significant, all groups had biologically significantly reduced body weight gains. Clinical signs of hypoactivity and half-closed eyes were also observed at 100 and 500 mg/kg/day and food consumption was lower than controls’ for females given 500 mg/kg/day.
There were no effects of treatment on pairing but a relationship between development of the fetuses and pups and treatment cannot be excluded because there was one female with only implantation scars and a dead fetus in the uterine horns and another female which delivered a litter of small pups which consequently died on day 3 post-partum.
Mean pup body weight at 500 mg/kg/day was less than that of the controls on day 1 post-partum and pup body weight gain was reduced at 100 and 500 mg/kg/day.
There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day, and the NOAEL for toxic effect on reproductive performance and on progeny was 100 mg/kg/day.

Executive summary:

Methods:

Three groups of ten male and ten female Sprague-Dawley rats received the test item, 2-EHN (CAS No. 27247-96-7), daily, by oral (gavage) administration, before mating and through mating and, for the females, through gestation until day 5 post-partum, at dose-levels of 20, 100 or 500 mg/kg/day. Another group of ten males and ten females received the vehicle, 0.5% tween 80 in purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 10 mL/kg.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals. The dams were allowed to litter and rear their progeny until day 6 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post-partum.

The males were sacrificed after completion of the mating period. The body weight, testes and epididymides weights were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. A microscopic examination was performed, for the control and high-dose group, on the testes and epididymides with special emphasis on the stages of spermatogenesis and histopathology of interstitial testicular structure.

The dams were sacrificed day 6 post-partum (or from day 25 post-coitum for females which did not deliver) and a macroscopic examination of the principal thoracic and abdominal organs was performed. In the females which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. A microscopic

examination was performed on the ovaries of the control and high-dose group females.

The pups were sacrificed on day 6 post-partum and were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

Results:

There were no animals prematurely sacrificed for reasons of poor clinical condition during the study.

All animals given 500 mg/kg/day had ptyalism throughout the study which, when measured, lasted 4-5 hours after dosing and may be related to the taste of the test item formulations. Hypoactivity, half-closed eyes and loud breathing were also observed sporadically in a few males and females. One female also had emaciated appearance, round back and piloerection for some days during the study. The majority of the females given 100 mg/kg/day had ptyalism during the premating and gestation periods and a few males had ptyalism during the premating period, which may be related to the taste of the test item formulations. One female at 100 mg/kg/day had hypoactivity and half-closed eyes on day 12 of dosing. There were no clinical signs at 20 mg/kg/day.

Male and female groups at all dose-levels gained less weight than the controls during the premating period in a dose-related manner achieving statistical significance at 500 mg/kg/day. Females given 500 mg/kg/day continued to gain less weight during gestation, however all groups had approximately comparable weight gains to the controls during lactation. Females given 500 mg/kg/day consumed less food than the controls throughout the study. This was not also observed in the males and there were no effects of treatment at the other dose-levels.

All pairs mated and the mean number of days taken to mate was comparable with the controls for all groups.

There were no effects on the numbers of corpora lutea or implantations. The mean numbers of pups born per litter were comparable with the controls at all dose-levels.

At 500 mg/kg/day, there was one female with one dead fetus and nine implantation scars in the uterine horns and one female which delivered a litter of small pups, all of which were dead by day 3 post-partum. As pup survival was higher in the control group, a relationship to treatment cannot be excluded.

Mean pup body weight at 500 mg/kg/day on day 1 post-partum was non-statistically significantly lower than that of the controls. Pup weight gain from day 1 to day 5 post-partum was non-statistically significantly lower than the controls’ at 100 and 500 mg/kg/day in a

dose-related manner, and mean pup body weights were non-statistically significantly lower than the controls’ on day 5 post-partum at both dose-levels.

There were no treatment-related pup clinical signs or necropsy findings.

There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Conclusion:

The test item, 2-EHN (CAS No. 27247-96-7), was administered daily by oral gavage to male and female Sprague-Dawley rats, from before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 20, 100 or 500 mg/kg/day.

Systemic toxicity was observed at all dose-levels, as evidenced by reduced body weight gain during, at least, the premating period (at least 13% less weight gained than the controls during the first 2 weeks of treatment) (statistically significant at 500 mg/kg/day). As body weight gains which are reduced by more than 10% are considered biologically significant, all groups had biologically significantly reduced body weight gains. Clinical signs of hypoactivity and half-closed eyes were also observed at 100 and 500 mg/kg/day and food consumption was lower than controls’ for females given 500 mg/kg/day.

There were no effects of treatment on pairing but a relationship between development of the fetuses and pups and treatment cannot be excluded because there was one female with only implantation scars and a dead fetus in the uterine horns and another female which delivered a litter of small pups which consequently died on day 3 post-partum.

Mean pup body weight at 500 mg/kg/day was less than that of the controls on day 1 post-partum and pup body weight gain was reduced at 100 and 500 mg/kg/day.

There were no treatment-related findings observed at macroscopic or microscopic examination of F0 animals and no effects on organ weights.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day, and the NOAEL for toxic effect on reproductive performance and on progeny was 100 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available Reproduction / Developmental Toxicity Screening Test on 2-EHN was conducted according to GLP and current OECD Test Guidelines at the time of study completion, and is assessed as Klimisch 1. However, additional data for this endpoint may be required based on the study results from the scheduled prenatal studies.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In addition to the OECD 421 study, a proposal was also submitted for reading-across to 2-EHN from a two-generation reproductive toxicity conducted using the source substance, Di-2 -Ethylhexyl Terephthalate (DEHT). This study was previously used in the registration dossier for 2-ethylhexan-1 -ol, a metabolite of 2 -EHN. A similar strategy, of reading across to 2 -ethylhexan-1 -ol was also used to meet the information requirements for several other endpoints for 2 -EHN. This read-across strategy has been rejected (Decision: TPE-D-0000002102 -92 -05/F). Therefore, the Toxicity to reproduction endpoint, and the data needed to meet current information requirements, will be re-evaluated following completion of the currently scheduled 90-day and prenatal studies and evaluation of the study results (see Sections on Repeated Dose Toxicity and Developmental Toxicity).

Effects on developmental toxicity

Description of key information

Several developmental toxicity studies in rodents are available for read-across substances 2-ethylhexan-1-ol, sodium nitrate and Pentaerythritol tetranitrate (PETN). No significant developmental effects were observed in these studies. No study is currently available in rabbits, and the requirement for a developmental study in a second species has been waived previously. However, this read-across strategy has been rejected (Decision: TPE-D-0000002102 -92 -05/F). Pre-natal developmental toxicity studies (according to OECD Test Guideline 414) with exposure to 2-EHN via the inhalation route are currently scheduled in rats and rabbits, according to Decision TPE-D-0000002102 -92 -05/F. The dossier will be updated with the study results once these become available.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experiment using the rapidly formed hydrolysis product of the target chemical conducted according to a protocol comparable to guideline OECD TG 414; publication meets generally accepted standards and is reported in sufficient detail

Justification for type of information:

Source substance, 2-ethyl-1-hexanol is the rapidly formed hydrolysis product of the target substance, 2-EHN.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

15 female test animals (instead of 25)

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, U.S.A.
- Age at study initiation: No data
- Weight at study initiation: 200-300 g at beginning of pregnancy
- Fasting period before study: No
- Housing: singly, in stainless steel mesh wire cages during gestation and exposure
- Diet: Ad libitum (during exposure-free periods)
- Water: Ad libitum (during exposure-free periods)
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±2
- Humidity (%): 50±10
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

Constant flow of test item mixed with a defined volume of heated air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hinners-type 0.5 m³ exposure chamber
- Method of holding animals in test chamber: In cages
- Source and rate of air: Compressed ambient air
- Method of conditioning air: Heating
- Temperature and humidity in air chamber: 77±2 F° (i.e. 25°C), 50±15 %
- Air flow rate: 0.5 m³/min
- Air change rate: 60/h

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly by infrared analyzer, twice a week by GC of charcoal tubes from the exposure chamber
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- A Miran 1A infrared analyzer was constantly used for hourly measurement.
- Additionally the Exposure concentrations were
verified weekly by a secondary analysis. Charcoal tube samples were drawn 2 days/week and analyzed by gas chromatography, with partial verification by samples spiked with known concentrations

Details on mating procedure:

- Proof of pregnancy: Sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

Gestation days 1-19

Frequency of treatment:

7 h every day

Duration of test:

19 days (dams were sacrificed on day 20)

Remarks:

Doses / Concentrations:
850 mg/m³ air
Basis:
analytical conc.

No. of animals per sex per dose:

15 (only females included)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Probably in expectation of absence of effects, the experimental equipment was used to provide the highest achievable concentration while keeping the mixing chamber temperature below 80 °F (ca. 27 °C)>

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Daily for the first week and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, calculated on a weekly basis

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Not reported
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes, all animals per litter
- Soft tissue examinations: Yes, half of the animals per litter
- Skeletal examinations: Yes, half of the animals per litter
- Head examinations: No

Statistics:

Multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA)

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: Overall feed consumption was approximatley 10-15% lower than in control animals.

Details on maternal toxic effects:
Although the weight gain appeared to be lower these difference did not reach statistical significance.

Dose descriptor:

NOAEC

Remarks:

Discriminating level = sole test concentration, 7 h/day, gd 1-19

Effect level:

>= 850 mg/m³ air

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 160 ppm

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 1 148 mg/m³ air

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 6.5 other: mmol/m³

Based on:

other: molar concentration of source and target chemical

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

Discriminating dose

Effect level:

>= 193 other: mg/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

Discriminating dose

Effect level:

>= 1.1 other: mmol/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No indication of malformations induced after exposure of pregnant rats were found.

Dose descriptor:

NOAEC

Effect level:

850 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: No toxicity to highest dose tested

Dose descriptor:

NOAEC

Effect level:

6.5 other: mmol/m3

Based on:

test mat.

Basis for effect level:

other: conversion to molar concentration to conver to 2-EHN concentration

Dose descriptor:

NOAEC

Effect level:

1 148 mg/m³ air

Based on:

act. ingr.

Basis for effect level:

other: conversion from molar concentration to 2-EHN concentration in mg/m3

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Summary of data (Litter mean ± standard deviation)

Data	Test item 2-EH	Control
Mean maternal weight [g]
Day 0	283 ± 18	243 ± 25
Day 7	286 ± 17	262 ± 24
Day 14	310 ± 17	291 ± 26
Day 20	371 ± 20	354 ± 32
Overall gain	88	111
Mean feed consumption [g]
Week 1	97 ± 17	108 ± 14
Week 2	107 ± 12	124 ± 12
Week 3	113 ± 11	118 ± 10
Overall mean	106 ± 15 (a)	117 ± 13
Mean water intake [g]
Week 1	196 ± 32	204 ± 46
Week 2	203 ± 24	276 t 93
Week 3	248 ± 30	265 ± 123
Overall mean	216 ± 36	248 ± 96
Mean corpora lutea per litter	15 ± 2	14 ± 4
Mean resorptions per litter	0.3	0.4
Mean number females per litter	7 ± 2	8 ± 2
Mean number males per litter	7 ± 2	7 ± 2
Mean fetal weight [g]
Female	3.02 ± 0.20	3.19 ± 0.20
Male	3.18 ± 0.30	3.28 ± 0.27

(a) Significantly different from control

Conclusions:

No maternal or foetal toxicity after inhalation exposure using the maximum vapour concentration of 850 mg/m³ air.

Executive summary:

The prenatal developmental toxicity of the source substance and test item 2-Ethylhexan-1-ol (CAS 104-76-7) via the inhalation route in the rat was measured in a GLP-compliant study using a “Prenatal Developmental Toxicity Study” compliant with OECD TG 414 (2001), with the sole deviation that the number of test animals was reduced (15 female test animals instead of 25). The validity criteria were met and the experiment can be considered relevant and adequate for the endpoint, however a reduced number of test animals were included. Nonetheless it is deemed conclusive and was rated „reliable with restrictions“, i.e. “Klimisch 2” according to the scale of Klimisch et al. (1997). In consideration of possible read-across from this study to metabolic precursors, additional uncertainties have to be taken into account. The rating of the study for use in such analogue or metabolite approach is lower compared to equally rated studies providing direct data from a target substance.

Fifteen sperm-positive female Sprague-Dawley rats were exposed to vapours of the test item through whole body exposure during gestation days 0 -19. Hinners type 0.5m³ exposure chambers to vapours of the test item at a concentration of 850 mg/m³ air was confirmed independently 2 methods; hourly by infrared analyzer and twice weekly by gas chromatography of charcoal tubes from the exposed chamber.

No maternal toxicity or developmental toxicity was noted in a rat inhalation study. The overall feed consumption was approximatley 10 -15% lower in treated animals compared to controls. Although the weight gain appeared to be lower in the treated animals, these differences did not reach statistical significance. The reproduction parameters were unchanged. No malformations were induced after exposure of the pregnant rats to the test substance. The incidences of resorptions, the number of fetuses per litter, the sex ratio, fetal weight, were not different to the control group and no external, skeletal or visceral malformations have been recorded.

In conclusion no substance-related adverse effects of the test item were observed at the highest vapour concentration achievable. Therefore the NOAEL (discriminating dose) was assigned to the highest test concentration. The NOAEL for maternal and developmental toxicity and teratogenicity in the rat is thus ≥ 850 mg/m³ air or ≥ 6.5 mmol/m³ as a basis for equimolar comparison in read-across approaches.

Reading-across to the target substance, 2-Ethylhexyl nitrate (2 -EHN, CAS 27247-96-7) delivers on the same molar concentration an air level of ≥1148 mg/m³ air. Taking the significantly lower vapour pressure of the target chemical (only 27 versus 93 Pa of 2 -EH at 20 °C) into account, an equimolar exposure exceeding saturated vapour concentrations of the target chemical can be concluded to be non-toxic during developmental exposure.

• Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally validated test guidelines, reported in sufficient detail; publication meets generally accepted standard

Justification for type of information:

The test substance, PETN has four nitrate groups that may be metabolized compared to one nitrate group on 2 -EHN. Therefore, this study represents a worst case scenario for the target substance, 2 -EHN.

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3550 Reproduction/Developmental Toxicity Screnning Test

GLP compliance:

not specified

Remarks:

No information is available on the GLP compliance status of this study.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA, U.S.A.
- Age at study initiation: 12 weeks
- Weight at study initiation: 200-300 g at beginning of pregnancy
- Fasting period before study: No
- Housing: singly, in stainless steel mesh wire cages during gestation and exposure
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 12-20
- Humidity (%): 30:70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dosing began 2 weeks prior to the mating period in both sexes and continued throughout the mating period. Males were dosed after the mating period until a total of 28 doses had been administered. Females were dosed continually throughout gestation and up to and including day 3 post-partum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A stock solution of 100 mg mg/ml PETN in corn oil was analyzed by gas chromatography using a flame ionization detector. The chromatography was done using a non polar column.

Details on mating procedure:

- Proof of pregnancy: Sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

56 days

Frequency of treatment:

daily

Duration of test:

56 days

Remarks:

Doses / Concentrations:
0 mg /kg bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
100 mg /kg bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
500 mg/kg bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, and more frequently when signs of toxicity are observed

BODY WEIGHT: Yes
- Time schedule for examinations: Male and female rats were weighed the day before and on the day of dosing, weekly thereafter and at termination of the study

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Not reported
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Not reported
- Number of late resorptions: Not reported

Fetal examinations:

- External examinations: Yes, all animals per litter

Statistics:

Bartlet's test for homogenicity of variance and a Kolmogorov-Smirnoff test for normality were used as a check for the analysis of varaince (ANOVA) assumptions.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no effects on maternal toxicity in any dose groups

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects at highest dose tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No indication of malformations induced after exposure of pregnant rats were found.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects at highest dose tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Since no effects on reproduction were observed, PETN is not considered to be a reproductive or developmental toxicant.

Executive summary:

Male and female sprague-dawley rats were exposed daily via oral gavage to varying concentrations (0, 100, 500, 1000 mg/kg bw/day) of PETN. Male rats were dosed 2 weeks prior to mating and throughout the mating period until a total of 28 doses had been administered. Female rats were dosed 2 weeks prior to mating, through the mating and gestation period and up to and including day 3 post-partum. Throughout the testing period general clinical observations were made at least once a day. No rats died following PETN exposure, however, four rats dies prior to scheduled euthanasia due to esophageal perforations caused by oral gavage. No differences were observed for gestation duration, number of pups produced and pup sex ratio. Two pups in the 100 mg/kg bw.day group and 1 runt from a control animals were found dead on day 2. No gross lesions were found for any of these pups. The only differences in body weight and feed consumption were observed relative to dose and not considered to be a direct cause of PETN toxicity.No differences in ovaryw eight (expressed as organ/ body weight ratios), number of implantation sites and number of corpora lutea were observed among treatment groups. Based on the absence of adverse effects from repeated daily exposure of up to 1000 mg/kg bw/day, PETN is not expected to be a reproductive or developmental toxicant.

PETN has four nitrate groups that may be metabolized compared to one nitrate group on 2 -EHN. Therefore, this study represents a worst case scenario for 2 -EHN. As no developmental effects were seen wtih PETN, no developmental effects are expected with 2 -EHN.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Read-across substance, 2-ethyl-1-hexanol is the rapidly formed hydrolysis product of the target substance, 2-EHN.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Effect level:

1 148 mg/m³ air

Based on:

other: conversion from source chemical to target chemical 2-ethylhexyl nitrate

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Effect level:

1 148 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: No toxicity to highest dose tested

Developmental effects observed:

no

Conclusions:

No maternal or foetal toxicity after inhalation exposure to the source substance using the maximum vapour concentration. Therefore, the target substance, 2-EHN can be concluded to be non-toxic during developmental exposure.

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

The test substance, PETN has four nitrate groups that may be metabolized compared to one nitrate group on 2 -EHN. Therefore, this study represents a worst case scenario for the target substance, 2 -EHN.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects at highest dose tested

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects at highest dose tested

Developmental effects observed:

no

Conclusions:

Based on the absence of adverse effects from repeated daily exposure of up to 1000 mg/kg bw/day, the source substance PETN is not expected to be a reproductive or developmental toxicant.

PETN has four nitrate groups that may be metabolized compared to one nitrate group on the target substance, 2 -EHN. Therefore, this study represents a worst case scenario for 2 -EHN. As no developmental effects were seen wtih PETN, no developmental effects are expected with 2 -EHN.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Available studies meet generally accepted standards and are reported in sufficient detail, but are on read-across substances. Therefore, the studies are considered to be Klimisch 2.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 148 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Available studies meet generally accepted standards and are reported in sufficient detail, but are on read-across substances. Therefore, the studies are considered to be Klimisch 2.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on results from the currently available studies, there is insufficient evidence to require classification of 2 -EHN for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008. Prenatal developmental toxicity studies with exposure to 2 -EHN via the inhalation route are currently scheduled in rats and rabbits, according to Decision TPE-D-0000002101 -92 -05/F. Once the study results are available, the dossier will be updated and this conclusion re-evaluated.