Dimethyl itaconate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test guideline (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-06-27 to 2014-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP; Relevant and adequate for this endpoint

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Remarks:

Double compliance with guidelines OECD 408 and 415

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Deviations:

no

Remarks:

Double compliance with guidelines OECD 408 and 415

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) approximately seven to eight weeks old
- Weight at study initiation: (P) Males: 193 - 245 g; Females: 144 - 180 g
- Fasting period before study: no
- Housing: prior pairing phase all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-07-19 to: 2012-11-25

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: formulations were prepared fortnightly and stored at 4ºC in the dark (the formulations to be stable for at least sixteen days).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis Oil was successfully used on the preliminary study and the same vehicle was therefore employed in this main study
- Concentration in vehicle: 0, 12.5, 25, 87.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of DMI in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.1 mg/mL. Standard solutions of test item were prepared in methanol at a nominal concentration of 0.1 mg/mL. For the homogeneity determinations, the 3.75 mg/mL test item formulations were assessed by visual inspection. The 250 mg/mL test item formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. The 3.75 mg/mL test item formulations were deemed homogeneous by visual inspection. The test item formulations were stable at 4° C in the dark for 16 days. The prepared formulations were within 5 % of the nominal concentration.

Duration of treatment / exposure:

approximately 17 weeks

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 50, 100 and 350 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

24 males and 24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a fourteen day dose range finding study, the investigation dosages of 500 and 750 mg/kg bw/day were associated with macroscopic stomach changes which were considered to preclude these dosages from any long term investigation of toxicity. No macroscopic changes were apparent in animals receiving 250 mg/kg bw/day during the fourteen day study. Therefore the low, intermediate and high dose levels were selected at 50, 100 and 350 mg/kg bw/day, respectively.

Positive control:

Not available

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily throughout the study (with the exception of the pairing phase).

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

The seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weights on days 1, 4, 7, 14 and 21 post partum, clinical condition of offspring from birth to Day 21 post partum.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed on Day 121 or Day 124
- Maternal animals: All surviving animals were killed on Day 21 post partum

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

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Colon, Caecum, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary tissue, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulating gland and fluids), Skin (hind limb), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach, Thyroid/parathyroid, Trachea, Testes, Tongue, Thymus, Urinary bladder, Uterus (with oviducts and cervix), Vagina.

ORGAN WEIGHTS
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicles (with coagulating gland and fluids), Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with oviducts and cervix)

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: the offsprings at interim deaths and the surviving offsprings until Day 21 post partum (terminal kill).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance: at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed usingBartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package.

Reproductive indices:

Pre-coital interval; fertility indices: mating index, pregnancy index; gestation length; parturition index; implantation loss: pre-implantation loss, post-implantation loss.

Offspring viability indices:

Live birth and viability indices

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient post-dosing salivation for both sexes at 350 and at 100 mg/kg bw/day. Similar postdosing salivation for one animal on a single occasion at 50 mg/kg bw/day. Females: 3 unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day.

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and food consumption of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance, fertility and gestation length were unaffected by treatment at 50, 100 or 350 mg/kg bw/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no statistically significant differences in organ weight that were considered to be of toxicological significance at 50, 100 or 350 mg/kg bw/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Diffuse epithelial hyperplasia of the non-glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day; this finding is adverse but represents a local irritant effect of the test item rather than systemic toxicity. No treatment related findings were observed at 50 or 100 mg/kg bw/day.

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at highest dose tested

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at highest dose tested

Dose descriptor:

NOEL

Remarks:

reproduction

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at highest dose tested

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Diffuse epithelial hyperplasia of the non-glandular region of the stomach for both sexes at 350 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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)
No effects

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
No effects

SEXUAL MATURATION (OFFSPRING)
Not examined

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
No effects

HISTOPATHOLOGY (OFFSPRING)
Not examined

Dose descriptor:

NOEL

Remarks:

survival, growth and development

Generation:

F1

Effect level:

>= 350 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at highest dose tested

Reproductive effects observed:

not specified

Conclusions:

Since treatment at 350 mg/kg bw/day was associated with adverse histopathological changes in the stomach for both sexes, the Lowest Adverse Effect Level (LOAEL) for local toxicity was considered at 350 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 350 mg/kg bw/day and the No Observed Effect Level (NOEL) was 100 mg/kg bw/day. The NOEL for reproduction and the survival, growth and development of the offspring was considered to be 350 mg/kg bw/day.

Executive summary:

The study was designed to investigate the effects of the test item on reproductive, pre-natal and post-natal development of the rat when administered through the reproductive cycle and to assess subchronic exposure of the test item to the rat. The study is considered to be compatible with both OECD TG 408 and 415.

The test item was administered by oral gavage to three groups, each of twenty-four male and twenty-four female Wistar Han™:RccHan™:WIST strain rats, for approximately seventeen weeks (and including for females 10 weeks pre-pairing, gestation and lactation phases) at dose levels of 50, 100 and 350 mg/kg bw/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, behavioural assessments, body weight change, food and water consumption and ophthalmic change were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group after 10 weeks of treatment, with females subsequently being allowed to litter and rear their offspring to Day 21 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights. During Week 10, extensive functional observations were performed on ten selected males and females from each dose group. Haematology and blood chemistry were also evaluated on ten selected males and females from each dose group. Adult males were terminated during Week 18 with surviving females and offspring on Day 21 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Following this assessment, histopathological evaluation of the stomach was extended to both sexes from the low and intermediate dosage groups. Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment. Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. No obvious neurological effects of treatment in behavioural assessment, in functional performance tests and sensory reactivity assessment were apparent at 50, 100 or 350 mg/kg bw/day. Body weight and body weight gain of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day. Food consumption of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day. Food conversion efficiency of both sexes was also unaffected at these dosages. At 350 mg/kg bw/day male water consumption was increased compared to control. Ophthalmic examinations at 350 mg/kg bw/day did not reveal any effect of treatment for either sex. Reproductive performance like mating, fertility and gestation length were unaffected by treatment at 50, 100 or 350 mg/kg bw/day. There was no effect of maternal treatment on corpora lutea and implantation counts, post-natal litter size, offspring survival or sex ratio, offspring body weight or body weight gain, litter weights or the clinical condition of the offspring at 50, 100 or 350 mg/kg bw/day. There were no treatment related effects on haematology parameters and on blood chemistry parameters for either sex at 50, 100 or 350 mg/kg bw/day. Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day. There were no statistically significant differences in organ weight that were considered to be of toxicological significance at 50, 100 or 350 mg/kg bw/day. Diffuse epithelial hyperplasia of the non glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day. No treatment related findings were observed at 50 or 100 mg/kg bw/day. In conclusion, treatment at 350 mg/kg bw/day was associated with local adverse histopathological changes in the stomach but this was considered to represent an irritant effect of the test item rather than systemic toxicity. Therefore the Lowest Observed Adverse Effect Level (LOAEL) for local toxicity was considered to be 350 mg/kg bw/day and the related NOAEL, local to be 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be ≥350 mg/kg bw/day. The NOEL for reproduction and the survival, growth and development of the offspring was considered to be ≥350 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Effect on fertility (oral): NOAEL actually ≥350 mg/kg bw/day, combined OECD TG 408/415 GLP-study (Fulcher & Watson 2014)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

- toxicity to reproduction: oral

In One Generation Reproduction Toxicity study in the rat (Fulcher & Watson, 2014) performed according to OECD TG 408 and 415 and under GLP the test substance was administered to rats via oral (gavage) route at 50, 100 and 350 mg/kg bw/day in Arachis oil vehicle. Each group (including control group) consisted of 24 males and 24 females. Diffuse epithelial hyperplasia of the non glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day. No treatment related findings were observed at 50 or 100 mg/kg bw/day. In conclusion, treatment at 350 mg/kg bw/day was associated with adverse histopathological changes in the stomach but this was considered to represent an irritant effect of the test item rather than systemic toxicity. Therefore the Lowest Observed Adverse Effect Level (LOAEL) for local toxicity was considered to be 350 mg/kg bw/day and the related NOAEL, local to be 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be ≥350 mg/kg bw/day. The NOEL for reproduction and the survival, growth and development of the offspring was considered to be ≥350 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Sole study for this endpoint

Effects on developmental toxicity

Description of key information

Assessed in a combined OECD 408/415 study covering a broader range of exposure and evaluation than required b OECD TG 414

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1 “Reliable without restrictions”, discriminating level (highest level tested)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Testing for this endpoint has been waived in accordance with Annex XI, sections 1.1.2. based on data not carried out according to the test methods referred to in Article 13(3). An experiment according to OECD TG 414 is considered scientifically unjustified.

As part of a test programme to evaluate the toxicity/hazard of DMI a study was conducted (Harlan Laboratories project number 41103744) that would allow for investigation of both Subchronic toxicity and Reproductive toxicity of the test item in the rat. The study was designed to ensure a more extensive evaluation of those parameters than that afforded by conduct of a combined repeat dose and reproduction screening test (OECD test Guideline 422). It was considered that the results of such a study would exceed the testing requirements of Annex VIII under REACH but may also be adequate, if the results of such a study support the premise, for Annex IX under REACH and therefore without the need for further animal testing. In particular it was felt that this single study will provide sufficient information to allow for the evaluation of effects on prenatal development without the necessity to use further animals. The rationale for not Conducting Further Animal Testing at Annex IX for Reproductive Toxicity Evaluation and the justification for not Conducting Prenatal Developmental Toxicity Study OECD 414 are given in detail in the waiving argument and bases on the expert statement of Wood (2015), attached in IUCLID section 13.

Justification for selection of Effect on developmental toxicity: via oral route:
Assessed in a combined OECD 408/415 study covering a broader range of exposure and evaluation than required b OECD TG 414; NOAEL reported under “Effects on fertility”

Justification for classification or non-classification

Based on the above stated assessment of reproduction toxicity potential of dimethyl itaconate, it could be concluded that the test substance does not need to be classified according to CLP (5th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.