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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline conducted under GLP. The study was selected as key because the information provided is sufficient for the purpose of hazard evaluation, classification and labelling, and risk assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
according to
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Germany
- Age at study initiation: approx 12-13 weeks
- Fasting period before study:no
- Housing: macralon cages type 3
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: approx 2 weeks

- Temperature (°C): 20 - 22
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
nose only
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: nose only cylindrical PVC column with a volume ~ 70 liters
- Method of holding animals in test chamber: cylindrical PVS column with a volume of ~ 70 liters surrounded by a transparent hood
- Source and rate of air: additional mass flow controlled stream of oxygen will be added to mid and high dose groups to provide sufficient and equal oxygen concentration to all groups
- Temperature, humidity, pressure in air chamber: 22-24 degrees Celcius, 30 - 63% humidity (means ~ 39%), oxygen concentrations 20.3 - 20.7%
- Air flow rate: 50 L/min

- Brief description of analytical method used:total carbon analysis with flame ionization detector
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
total carbon analysis with flame ionization detector
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/2
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not necessary
- Verification of same strain and source of both sexes: all animals were same strain, maintained in a SPF colony from a reputable supplier
- Proof of pregnancy: [ sperm in vaginal smear] referred to as [day 0 ) of pregnancy
Duration of treatment / exposure:
gestation day 6 - 19
Frequency of treatment:
6 hours/day
No. of animals per sex per dose:
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:high dose same as used for repeated dose toxicity studies. Concentrations higher than 50,000 ppm can result in secondary effects due to oxygen deprivation.


Maternal examinations:
- Time schedule: daily

- Time schedule for examinations: GD 0, 3, 6 , 9, 12, 15, 19, 21

- Sacrifice on gestation day # 21
- Organs examined: Gross necropsy with tissues showing macroscopic abnormalities removed and stored for future histological examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- Soft tissue examinations: Yes: half per litter
Skeletal examinations: Yes: half per litter
Fisher exact probability test - clinical findings, number of mated and pregnant females, number of females with live fetuses, sex ratio, fetopathological screening; ANOVA followed by Dunnett's multiple comparison test - body weight (gain), organ weights, food consumption; Kruskal-Wallis nonparametric analysis of variance followed by Mann-Whitney U test - number of corpora lutea, implantation sites, pre and post implantation loss, live and dead fetuses, and early and late resorptions. Litter basis was used for fetuses. All analysis were 2 sided. Group mean differences with an associated probability of less than 0.05 were considered statistically significant.
female fecundity index, gestation index,
Historical control data:
not included in report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: no treatment related adverse effects see details below

Details on maternal toxic effects:
During the course of the study, there were variations in body weight, body weight gain and food consumption. These effects were seen primarily during exposure days 6-9 but not evident when data evaluated for days 0 - 21 or 6 - 19. However, overall these differences did not follow an exposure related pattern and ultimately viewed over the entire period of gestation did not appear to represent an adverse effect. Pregnant females (24, 21,24,25) had live fetuses at Caesarean section in the control, low, mid and high dose group. No differences were observed in number of implantation sites, pre/post implantation loss, live or dead fetuses, resorptions, or sex ratio, female fucundity index or gestation index between the groups. No treatment related effects were noted in weight of gravid uterus, empty uterus or ovaries. Macroscopic findings did not reveal any remarkable or treatment related findings. Transient test substance-related and statistically significant effects on body weigh changes at the onset of the exposure period had no impact on either final body weight or on cumulative body weight gain at any exposure level tested.

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
ca. 50 000 ppm
Based on:
test mat.
Basis for effect level:
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: delayed ossification and wavy ribs effects observed in all dose groups and not considered to be adverse

Details on embryotoxic / teratogenic effects:
There were no difference in number and percent of live offspring, sex ratio, fetal weight, fetal visceral anomalies, variations, incidentals between groups. Statistically significant changes in small and large fetuses were observed. However, these effects did not follow a dose response and fetal weights were not significantly different from controls. No statistically significant differences in the incidence of skeletal malformation were observed between groups. An increase in fetal skeletal variations was noted in the low and high dose group, this effect was primarily due to an increase in irregular shape of one sternebra in the low dose group; these increases were not dose dependent. There were increases in the frequency of wavy ribs in all treatment groups (see attached table for details). These observations are typically reversible and in the case of the current substance, the reversibility of this finding was demonstrated in a subsequent two-generation reproduction study. The overall incidence for variation in ossification was 100% in all control and all test material exposed fetuses (see attached table). However, it has been established (Carney and Kimmel, 2007 - see attached) that in rats, wavy ribs and delayed ossification are not considered adverse events in the absence of any other evidence of adverse developmental toxicity.

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
50 000 ppm
Based on:
test mat.
Basis for effect level:
other: No treatment related adverse effects were observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

It was concluded no adverse maternal and developmental effects were observed in rats after inhalation for 6 hours a day with test substance during GD 6-19 at a concentration up to 50000 ppm (233000 mg/m3). These conclusions are drawn from this study which meets the requirements of OECD guideline 414.
Executive summary:

There was no evidence of test substance-related maternal toxicity at any concentration tested; maternal effects were limited to non-adverse and transient body weight reductions at the onset of dosing which had no impact on either final weights or cumulative weight gains. Fetal effects were limited to non-adverse increases in wavy ribs and delayed ossification. These effects were not observed when selected pups in the 2 generation reproduction study were evaluated. Therefore the fetal and maternal NOAEL is 50000 ppm (233000 mg/m3), the highest level tested.