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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.10.2006 to 21.12.2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Only one concentration tested.
GLP compliance:
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: (P) Males: 300.7-350 g; Females: 195.5-240 g
- Fasting period before study: No
- Housing: Individually housed in suspended wire-mesh cages. Except during mating, gestation and lactation periods. Mating: home cage of the male; Gestation: shoebox cages; Lactation: dams housed with litters.
- Diet (e.g. ad libitum): Ad libitum (except during inhalation exposure or FOB.
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Five days

- Temperature (°C): 20.95-21.71
- Humidity (%): 31-69
- Air changes (per hr): 12.1
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27.10.2006 To: 21.12.2007

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
clean air
Details on exposure:
- Exposure apparatus: 1000 litre stainless steel and glass, TSE-system style, whole-body inhalation exposure chambers.
- Method of holding animals in test chamber: In cages
- Source and rate of air: Nash Air Compressor (rate not specified).
- Method of conditioning air: Series of filters to remove contaminants.
- Temperature, humidity, pressure in air chamber: Temp: 19-25oC (no other information, but it was stated that conditions were maintained according to the protocol).
- Air change rate: No data
- Treatment of exhaust air: No data

- Brief description of analytical method used: No data
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Continuous until evidence of copulation observed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Shoebox cages.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
None given (apparent Annex missing)
Duration of treatment / exposure:
Males: 30 days
Toxicity group females: 29 days
Reproductive group females: 15 days prior to mating, through the mating period and up to day 19 of gestation.
Frequency of treatment:
Daily (seven days per week)
Doses / concentrations
Dose / conc.:
5.1 mg/L air
target concentration equivalent to 400 ppm
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previously conducted study.


Parental animals: Observations and examinations:
- Time schedule: All animals were observed at least twice daily in their cages for mortality, morbidity and moribundity throughout the in-life phase of the study. General clinical observations were made at least once per day, beginning on the first day of treatment (except on days of detailed examinations). Clinical observations were also performed on all animals on the day of, but prior to, scheduled necropsy.

- Time schedule: All animals, once before the first exposure and weekly thereafter. Examinations included but were not limited to changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movement, stereotypies, difficult or prolonged parturition or bizarre behaviour.

- Time schedule for examinations: Individual body weights were determined beginning with randomisation into test groups, on the first day of exposure, at least weekly thereafter, and the day of necropsy. During gestation, the females were weighed (at a minimum) on gestation days 0, 7, 14 and 20 within 24 hours after parturition, and day 4 postpartum.

FOOD CONSUMPTION: Individual animal food consumption was recorded at least weekly on an individual animal basis for the periods listed: Male rats: two week pre-mating period only (feeder weights were taken on days 1, 8 and 15). Female rats: toxicity group on day 1 of exposure to necropsy (feeder weights were taken on day 1, 8, 15, 22 and the day prior to necropsy. Reproductive female rats: two week pre-mating period, gestation and postpartum (feeder weights were taken on days 1, 8, 15 and on gestation days 0, 7, 14, 20 and on day 0 and 4 postpartum).


OTHER: The duration of gestation was calculated from Day 0 gestation for each female. From Day 20 after evidence of mating, pregnant animals were checked at least three times daily for evidence of parturition.

Functional Observational Battery (FOB) performed on all adult males and all toxicity group females prior to the start of exposure and during the fourth week of exposure (prior to daily exposures).

Clinical pathology assessments on all adult male and toxicity group females - See section 7.5.2.
Oestrous cyclicity (parental animals):
No evidence that estrous cyclicity was investigated.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight and epididymis weight.
Litter observations:
- Performed on day 4 postpartum: no

The following parameters were examined in F1 pups: each litter was examined as soon as possible after delivery to determine the number and sex of the pups, the number of live pups, number of pups dead, runts. Live pups were counted, sexed and the sex ratio calculated. Litter weights were taken within 24 hours of parturition and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities. Possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
- Male animals: All surviving animals after 30 days exposure.
- Maternal animals: Day 4 postpartum.

- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. For pregnant females, the number of corpora lutea and the number of implantation sites were recorded. For the three females with positive evidence of mating that failed to deliver a litter, the uterus was stained to enable counting of possible reabsorbed implant sites.

HISTOPATHOLOGY / ORGAN WEIGHTS: At necropsy, the following organs from males and toxicity group females were weighed: adrenal glands, brain, heart, lungs, kidneys, liver, spleen and thymus. Testes, epididymides, seminal vesicles and prostate weights were recorded for all male adult animals. Ovaries with oviducts and uterine weights were recorded for toxicity group females. Selected organs and tissues were examined histopathologically in the toxicity group males and females, not reproductive group females (see Section 7.5.2).
Postmortem examinations (offspring):
SACRIFICE: Day 4 post-partum.

GROSS NECROPSY: Dead and sacrificed pups examined for external gross abnormalities only.

All data analyses was conducted using SAS version 9.1.3. Statistically significant probabilities were reported for p-values of <0.05, <0.02 and <0.01.
Reproductive indices:
Gestation length, mean number of implantation sites, mean number of corpora lutea, mean mating and fertility indices. Mean litter size, mean live litter size, mean litter weight, mean ratio live births/litter size.
Offspring viability indices:
Survival to postpartum day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No deaths and no significant treatment-related clinical signs of toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Significant increases in body weight gains were noted in the 400 ppm parental females during the third week of gestation, which were not considered to be treatment-related. There were no statistically significant differences in food consumption in females. The food consumption for treated males was significantly decreased during weeks 1 and 2 and for total food consumption. However, food consumption for weeks 1 and 2 was within the normal range of the laboratory's historical controls. The difference was not considered to be related to treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): there was no effect on testes or epididymides weights. No other parameters were examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Three female rats in the 400 ppm group with evidence of copulation failed to deliver a litter. One of these three females showed signs of parturition (blood discharge) on gestation day 25, but no pups were found. However, seven implant sites were present. The remaining females produced litters that were similar to the controls.

There were no treatment-related effects apparent for any of the reproductive endpoints. There were no changes observed in the litter size, male-to-female ratio, pup body weights or the pup survival.

ORGAN WEIGHTS (PARENTAL ANIMALS): There were no differences in absolute organ weights. The spleen to body weight ratio was slightly lower for toxicity group females, but not males, exposed to 400 ppm test substance. There was no histopathological correlate, nor any effect of exposure in other lymphoid tissues. This was considered to be random variation and not of toxicological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS): There were no gross lesions attributed to the test substance.

HISTOPATHOLOGY (PARENTAL ANIMALS): One possible exposure-related finding was increased minimal alveolar histiocytosis in males in the 400 ppm exposure group. This is a common non-specific finding in inhalation studies; however, the minimal severity and fact that only one sex was affected made the significance of the finding uncertain. There were no other exposure-related in other tissues.

FOB: There appeared to be no functional or neurological effects of the test substance on the rats.

Effect levels (P0)

Key result
Dose descriptor:
Effect level:
>= 400 ppm
Based on:
test mat.
Basis for effect level:
other: No significant treatment-related effects on males or toxicity phase females.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

There were no adverse effects for the pups up to postpartum day 4.

Effect levels (F1)

Key result
Dose descriptor:
Effect level:
>= 400 ppm
Based on:
test mat.
Basis for effect level:
other: No adverse effects on pups.

Overall reproductive toxicity

Reproductive effects observed:

Applicant's summary and conclusion

In a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted using a protocol comparable to OECD 422 and to GLP (reliability score 1) the NOAEC for general and reproductive toxicity was at least 400 ppm (the only concentration tested) according to the study report. However, it should be noted that three female rats in the 400 ppm group with evidence of copulation failed to deliver a litter. One of these three females showed signs of parturition (blood discharge) on gestation day 25, but no pups were found. However, seven implant sites were present. The remaining females produced litters that were similar to the controls.