Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2016 - May 2017 (in life)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(July 2016)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Stability of the test substance in the solvent/vehicle: Formulations of 10 to 100 mg/mL were found stable and homogenous for 7 days at room temperature (Currenta report no. 2013/0091/12).

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han)
- Source: Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (at the start of dosing)
- Weight at study initiation: Males: 313.1-407.0 g; Females: 193.6-262.1 g (at the start of dosing)
- Housing: during the pre-pairing phase in groups of up to four; following mating females were housed individually during gestation and with their litter during lactation.
- Diet and water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried with molecular sieve

Details on exposure:

A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weights.

PREPARATION AND HANDLING OF DOSING SOLUTIONS:
Formulations were prepared weekly. The test article was formulated as a suspension in dried Corn Oil following dispensary SOPs. Formulations were stored at room temperature (15 to 25 °C) in a sealed container, protected from light. Formulations were stirred continuously (excluding controls) from at least 30 minutes before commencement of, and throughout, dosing.

Details on mating procedure:

Animals were paired on Day 15 of study (Pairing Day 0). During the pairing phase, one male was housed for up to 10 days with one female of the same dose group. Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon confirmation of mating, vaginal washing was discontinued, and the male was removed. The day on which mating was confirmed was designated GD 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (1 x 30 mL [random] aliquot from test article and control article [vehicle] formulations) prepared for use on Day 1 and during Week 6 were taken for achieved concentration analysis. The analysed formulations met requirements and were within the limits of analytical accuracy of measurement.

Duration of treatment / exposure:

Males: up to 62 days (2 weeks prior to pairing, during the pairing phase, and until the day before necropsy)
Females: up to 57 days (weeks prior to pairing, during the pairing phase, and until Lactation Day 13, inclusive; one female was not dosed on Gestation Day 21 as the animal was in parturition)

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 1000 mg/kg was chosen as the high dose since this dose level did not show any overt toxicity based on a previous range finding study (Study no. 8350179). Using a two fold interval, the intermediate and low dose levels 500 and 250 mg/kg, respectively, were chosen as appropriate.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity. Animals were observed daily for the first 3 days of dosing; upon return to the home cage; and approximately 0.5, 1, 2, and 4 hours postdose.
With respect to natural delivery: Animals were observed three times each day at the beginning, middle, and end of the day, starting when the first females reached GD 21 and until the last female had littered. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked the end of gestation; where not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to LD 1 observations. Abnormal signs of nesting, parturition, or nursing were recorded. Special care was taken to disturb the dam as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record of the clinical condition of each animal was maintained.

BODY WEIGHT: Yes
Male body weights were recorded once during acclimation, on the first day of dosing, and at weekly intervals thereafter. Female body weights were recorded twice during acclimation, on the first day of dosing, weekly prior to pairing; until confirmation of mating; on GD 0, 7, 14, and 20; and on LD 1, 4, 13, and 14 (prior to necropsy).

FOOD CONSUMPTION: Yes
The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 1 to 13. Consumption was calculated as g/animal/day.

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

During acclimation, daily vaginal washings were taken from all females from 1 week after arrival until the day prior to dosing; the stage of estrous was recorded, and only females with regular 4 to 5 day cycles were included on study. Daily vaginal washings were taken from females from the start of dosing until the confirmation of mating and on LD 14.

Sperm parameters (parental animals):

not performed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On PND 4, litters were culled to 10 pups/litter (five pups/sex where possible). Runts (pups considered unlikely to survive to weaning) were pre selected for cull. The remaining pups were selected randomly. Culled pups were examined macroscopically, and pooled blood samples were taken for thyroid hormone assessment. Culling was considered to have created a uniformed litter size, which reduced differences in pup body weight due to litter size.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter sizes (stillbirths, live births) and pup sex were recorded on PND 1, 4, 7, and 13. Daily records of mortality and changes in litter sizes were maintained. Each pup was given a detailed physical examination from PND 1. Individual pup body weights were recorded on PND 1, 4, 7, and 13. Ano-genital distance was recorded on PND 4. The number of nipples/areolae for male pups was counted on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where possible, pups found dead or in a moribund condition were examined macroscopically.

Postmortem examinations (parental animals):

SACRIFICE
Females were sacrificed in a controlled randomization sequence on Lactation Day 14. Males were sacrificed in a controlled randomization sequence on Post-Pairing Day 42 (Day 63 of study).

CLINICAL PATHOLOGY
Blood samples of adult animals for thyroid hormone analysis (2 x 0.6 mL) were withdrawn from the abdominal aorta at necropsy; sampling was performed at a similar time on each occasion.

GROSS NECROPSY
All animals were subjected to necropsy. After sacrifices, macroscopic examinations were conducted, and all lesions were recorded. The uterus of each female was immersed in a 10 % ammonium sulphide solution, and implantation sites were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were recorded for each animal of the F0 generation, excluding decedents. Bilateral organs were weighed together. Tissues dissected were freed from fat and contiguous tissue.
List of organ weights determined: Cowper’s glands (bulbo-urethral gland), epididymis, kidney, levator ani plus bulbocavernosus muscle complex, liver, ovary, prostate, seminal vesicle (with coagulating glands), testis (including Tunica albuginea), thyroid and parathyroid, uterus (including cervix).

The following tissues from the F0 generation of control and high dose group and of all unscheduled deaths/sacrifices were examined (preservation in 10 % neutral buffered formalin/staining with hematoxylin and eosin, unless otherwise indicated): epididymis (Modified Davidsons fixative, also stained with PAS), gross lesions, kidney, liver, ovary, stomach, testis (including Tunica albuginea)(also stained with PAS). Slides of the testis and epididymis from the first five control and high-dose F0 generation males were examined by the Study Pathologist; for all other tissues all ten animals/sex and control or high dose group were used for microscopical assessment. Additionally, the liver from F0 generation animals in low and mid dose animals were examined.

Postmortem examinations (offspring):

SACRIFICE
Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose).

CLINICAL PATHOLOGY
Pups culled on PND 4 had blood samples (1 x 0.6 mL) for thyroid hormone analysis withdrawn via decapitation to provide one pooled sample for each litter, where possible. Pups sacrificed on PND 13 had blood samples (2 x 0.6 mL) for thyroid hormone analysis withdrawn by cardiac puncture to provide two pooled samples for each litter (one sample from two males and one sample from two females, where possible).
Total T4 and TSH were determined for Thyroid Hormone Analysis.

GROSS NECROPSY
After termination, full macroscopic examinations were conducted for all decedents, surplus pups culled on PND 4, and pups sacrificed on PND 13, paying particular attention to the external reproductive genitals; all lesions were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid was removed from one pup/sex/litter on PND 13 and weighed approximately 24 hours post fixation. The thyroid was microscopically examined.

Statistics:

Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Body weight, body weight gain, food consumption (gestation and lactation phases), absolute organ weights, organ : terminal body weight ratios, terminal body weights, and PND 13 pup thyroid hormone data only (male, female, and combined) were analysed using analysis of variance (ANOVA).
Male and female mating, fecundity, and fertility indices were analysed using a one sided lower tail Fisher’s exact test.
Mean number of estrous cycles and mean cycle length was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Percent pregnant, percent delivering, and gestation index were analysed using a one sided lower tail Fisher’s exact test.
Percent of females with stillborn pups was analysed using a one-sided upper tail Fisher’s exact test.
The duration of gestation; number of implantation sites; number of pups born; number of pups alive on PND 1, 4 (before and after culling), 7, and 13; number of pups culled on PND 4; percent post-implantation loss; and live birth and survival indices were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Pup body weights (male, female, and combined) were analysed using analysis of covariance (ANCOVA), with litter size as the covariate.
Live pups/litters with live pups were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Ano-genital distance (male pups only) were analysed using analysis of covariance (ANCOVA), with litter size as the covariate

Reproductive indices:

Fertility and pregnancy indices (Mating Index, Female and Male Fecundity Index, Female and Male Fertility Index, % Pre-implantation Loss, % Post-implantation Loss, % male pups, Gestation index, % post implantation survival index, % live birth index

Offspring viability indices:

Survival indices 1-4, 4-7, and 7-13

Description (incidence and severity):

Rapid respiration and vocalization were noted for some animals. Only a few incidences of these observations were noted, and no dose relationship occurred; as such, their relation to administration with the test article was unclear.
Dose-proportional increased incidences of soft/loose feces and piloerection were noted for males, with increased incidences of wet fur and fur staining also noted. These observations were correlated with a reduction in body weight gain. These observations, while considered due to administration of the test substance, were considered not adverse.
A few incidences of mouth rubbing were noted for test article-treated animals; this is a common observation in oral gavage administration and is associated with the unpalatable test articles. The finding was considered non-adverse.

Description (incidence):

One control male was found dead on Pre-Pairing Day 8. Macroscopic observations indicated the animal was mal-dosed.
One female administered 500 mg/kg/day was found dead on LD 7. The dam was not observed to have clinical observations prior to death, and no macroscopic finding was noted, therefore the death is unexplained.

Description (incidence and severity):

A reduction in mean body weight gain was noted between Pre-Pairing Day 1 and 8 for males administered 1000 mg/kg/day. Other changes in body weight gain were believed to be attributed to the use of corn oil as a control article (vehicle) and not related to administration of the test substance.

Description (incidence and severity):

Control animals were noted to have reduced food consumption, compared to background control values. This observation is often noted in animals administered corn oil as a vehicle control article.

Description (incidence and severity):

Males administered 1000 mg/kg/day had a statistically significant increase in TSH, because one outlying male (animal 34) had a markedly higher value. All other values were similar to background control ranges.

Description (incidence and severity):

A minor hepatocellular hypertrophy/eosinophilia (Grade 1) recorded in the liver of few F0 generation animals administered 250, 500 or 1000 mg/kg/day was attributed to hepatic enzyme induction. However, since one male animal administered 1000 mg/kg/day revealed a higher severity in this finding (i.e. Grade 2) 1000 mg/kg/day might constitute a first indication of an adverse liver effect.
No other test article-related microscopic findings were recorded.

Description (incidence and severity):

No effect on estrous cycles was noted.

Description (incidence and severity):

No effect on mating, fertility, or pregnancy was noted (i.e. in all treated groups mating, fecundity and fertility indices were found to be 100 %).

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on a temporary reduction in mean body weight gain in males and a first indication of an adverse liver effect at 1000 mg/kg and day.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity/fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproductive performance/fertility observed

Critical effects observed:

no

Description (incidence and severity):

No adverse clinical observations were noted.

Description (incidence and severity):

Five pups were found dead, sacrificed, or recorded as missing (presumed cannibalized) between PND 0 and 13. These were distributed evenly between the groups, including control, and no specific cause of demise was determined for these animals. Their deaths are considered to be incidental and not due to administration of the test article.

Description (incidence and severity):

A minor increase in body weights was noted on PND 1 and 4 for litters of dams administered 250 or 500 mg/kg/day. However, by PND 13, an overall slightly reduced body weight gain was noted for litters of dams administered 1000 mg/kg/day, with pups (both sexes) observed to have slightly lower recorded body weights than control litters, combined values were 93 % of control values. The overall body weight gain for pups of litters administered 1000 mg/kg/day was 90 % of control. This effect on bodyweight gain is considered to be attributed to administration of test article to the dams.

Description (incidence and severity):

Thyroid hormone analysis revealed comparable values as controls.

Description (incidence and severity):

No noticeable effects on pub thyroid weight detected.

Description (incidence and severity):

No macroscopic findings considered related to test substance administration to adults were recorded in the surplus offspring pups of both sexes sacrificed on Post-Natal Day 4.
No macroscopic findings considered related to test substance administration to adults were recorded in the offspring pups of both sexes sacrificed on Post-Natal Day 13.

Description (incidence and severity):

The mean percentage of male pups on PND 1 was statistically significantly higher for litters of dams administered 250 mg/kg/day. This was not noted in higher dose groups; as such, the observation was considered incidental.
No effect on anogenital distance was noted.
Nipple/Areolae Count: None were noted.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

The no observed adverse effect level (NOAEL) for this study is considered 1000 mg/kg/day.

Reproductive effects observed:

no

No specific target organ toxicity was revealed after oral administration of the test article up to and including 1000 mg/kg/day. Administration of 1000 mg/kg/day led for both F0-animals and the offspring to a slight effect on body weight gain. A minor hepatocellular hypertrophy/ eosinophilia was attributed to hepatic enzyme induction.

Due to a temporary reduction in mean body weight gain in males and a first indication of an adverse liver effect the NOAEL for the F0 generation is considered 500 mg/kg/day. The NOAEL for reproduction/fertility is 1000 mg/kg/day.

Due to the effects on body weight and bodyweight gain the NOAEL for offspring development is considered 500 mg/kg/day, based on the reduction in body weight gain noted on PND 13.

Executive summary:

For assessment on reproductive toxicity a screening for reproductive / developmental toxicity, according to OECD TG 421, was conducted. In this study, four groups of 10 male and 10 female Wistar rats were daily administered 0 (vehicle control), 250, 500, or 1000 mg/kg test substance in dried corn oil by gavage (administration volume 10 mL/kg). Males were treated for 62 days and females for up to 57 days; this included a two-week pre-pairing period and the mating phase, and the period before necropsy. Females were dosed up to lactation day 13 inclusive.

No specific target organ toxicity was revealed after oral administration of the test article up to and including 1000 mg/kg/day. Administration of 1000 mg/kg/day led for both F0-animals and the offspring to a slight effect on body weight gain. A minor hepatocellular hypertrophy/ eosinophilia was attributed to hepatic enzyme induction.

Due to a temporary reduction in mean body weight gain in males and a first indication of an adverse liver effect the No Observed Adverse Effect Level (NOAEL) for the F0 generation is considered 500 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for reproduction/fertility is 1000 mg/kg/day.

Due to the effects on body weight and bodyweight gain the NOAEL for offspring development is considered 500 mg/kg/day, based on the reduction in body weight gain noted on PND 13.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For assessment on reproductive toxicity a screening for reproductive / developmental toxicity, according to OECD TG 421, was conducted. In this study, four groups of 10 male and 10 female Wistar rats were daily administered 0 (vehicle control), 250, 500, or 1000 mg/kg test substance in dried corn oil by gavage (administration volume 10 mL/kg). Males were treated for 62 days and females for up to 57 days; this included a two-week pre-pairing period and the mating phase, and the period before necropsy. Females were dosed up to lactation day 13 inclusive.

No specific target organ toxicity was revealed after oral administration of the test article up to and including 1000 mg/kg/day. Administration of 1000 mg/kg/day led for both F0-animals and the offspring to a slight effect on body weight gain. A minor hepatocellular hypertrophy/eosinophilia was attributed to hepatic enzyme induction.

Due to a temporary reduction in mean body weight gain in males and a first indication of an adverse liver effect the NOAEL for the F0 generation is considered 500 mg/kg/day. The NOAEL for reproduction/fertility is 1000 mg/kg/day.

Due to the effects on body weight and bodyweight gain the NOAEL for offspring development is considered 500 mg/kg/day, based on the reduction in body weight gain noted on PND 13.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The available screening for reproductive / developmental toxicity (OECD TG 421) reveals at 1000 mg/kg/day on PND 13 a slight reduction in body weight gain for the offspring. This is considered a secondary effect to the slight general toxicity of the substance to the dams; at 1000 mg/kg/day also a slight effect on body weight gain and a first indication of a beginning liver toxicity was seen for the parental animals.

No other findings were recorded for the offspring, thus the oral NOAEL for developmental toxicity as well as for general toxicity is considered 500 mg/kg.

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, Annex I, classification for toxicity to reproduction is not justified.

This is because the available reproductive toxicity screening study reveals no indication for reproductive toxicity or a primary developmental toxic effect. Furthermore, no indication of systemic toxicity at all is observed in any of the available acute and repeated toxicity studies.