Amines, C12-14-alkyldimethyl

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study (OECD 422) and according to GLP. Justification for read-across see chemical safety report chapter 1.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

As not every positive mating sign results in pregnancy, the mating period was extended until a positive mating sign was noted for all females (up to 17 days). This additional mating period was conducted to guarantee at least 8 pregnant females per group

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemical Law and OECD Principles of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-18-alkyldimethyl, N-oxides

EC Number:

273-281-2

EC Name:

Amines, C12-18-alkyldimethyl, N-oxides

Cas Number:

68955-55-5

IUPAC Name:

dimethyl(pentadecyl)amine oxide

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: Males 327.9 - 424.0 g; females 212.7 - 280.0 g
- Housing: Except during the mating period, the animals were kept singly in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm.
- Diet: Ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 3°C (maximum range)
- Humidity (%): Relative humidity of 55% - 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle (tap water) to the appropriate concentrations and was administered orally at a constant application volume of 10 mL/kg b.w./day. The test item-diet mixture was freshly prepared every day.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg b.w./day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At study initiation:
Analysis of stability and concentration: Immediately after preparation of the mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 9.
Analysis of Homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). Total number of samples: 9.

At study termination:
Analysis of concentration: During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). Total number of samples: 3.
Test item-vehicle mixtures (in total 21 vials), 4 further vials containing the test item or vehicle of test days 1 and 42 were dispatched on dry ice by courier for analysis.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks, if a positive mating sign was not observed an additional mating period was carried out with the same partneruntil a positive mating sign was noted for all females
- Proof of pregnancy:[vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single

Duration of treatment / exposure:

Main study males (mated):
50 % of the main study males were dosed for 31 days and the other 50 % of the main study males were dosed for 36 days, depending if they were sacrificed on test day 32 or 37. This dosing period includes the pre-mating period (2 weeks), the mating period (1 to 17 days) and the post mating period up to and including the day before sacrifice (terminal sacrifice was conducted on test day 32 or 37.

Main study females (mated):
The main study females were dosed for 41 to 56 days. This period includes 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Satellite animals (not mated animals):
The satellite animals were treated for 41 days followed by a recovery period of 16 days. The animals were not mated. Treatment started on the same day as of the main study animals and was conducted up to the day before the first scheduled sacrifice of the main study dams on test day 42.

Frequency of treatment:

once daily

Duration of test:

males: 32-37 days
females: 41-56 days

No. of animals per sex per dose:

TThe animals were randomly allocated to the 4 test groups as follows:

Group test substance No. and sex
dose of animals
[mg/kg b.w./day]# MS+RP

1 0 10 m + 5 m
(control) 10 f + 5 f

2 40
(low dose) 10 m 10 f

3 100
(intermediate dose) 10 m 10 f

4 200 10 m + 5 m
(high dose) 10 f + 5 f

MS: main study; animals scheduled for the reproduction study
RP: recovery period; satellite animals scheduled for a 16-day recovery period, the
animals were not mated and thus not used for the reproduction study
m: males
f: females
#: A correction factor of 3.13 was used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available toxicological data and a preliminary dose-range finding study (LPT study no. 25122). No mortality was noted in this dose-range-finding study no. 25122. Signs of systemic intolerance were noted in the animals of both sexes starting at 40 mg Aromox B-W 500/kg b.w./day. A slightly to severely reduced mean food consumption and mean body weight were noted in the male rats starting at 100 mg/kg b.w./day or at 250 mg/kg b.w./day, respectively. The females were not affected.
Necropsy revealed a thickened cardiac region of the stomach in one male rat treated with 250 mg/kg b.w./day as well as in all high dosed male and female rats treated with 400 mg Aromox B-W 500/kg b.w./day.

- Rationale for animal assignment: Random. At commencement of the study, the weight variation of animals used was minimal and did not exceed 20% of the mean weight of each sex.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.
- Cage side observations included: Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality. Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals. Detailed clinical observations were made in all male main study animals until terminal sacrifice in test week 5 or 6, respectively, in all female main study animals until the day of parturition and in all male and female satellite animals until terminal sacrifice in test week 8. These observations were made outside the home cage in a standard arena at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Mortality: Further checks were made early in the morning and again in the afternoon of each working day to identify dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily throughout the test period.

BODY WEIGHT: Yes
- Time schedule for examinations: females were weighted on the first day of dosing, weekly thereafter and at study termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation. Body weights were recorded individually for each adult animal.

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

The test item-treated groups 2 to 4 were compared with the control group 1.
STUDENT's t-test: All numerical functional tests (p ≤ 0.01)
The following limits were used:
p ≤ 0.01 ≙ t = 2.878 (18 degrees of freedom)
p ≤ 0.01 ≙ t = 2.921 (16 degrees of freedom)
p ≤ 0.01 ≙ t = 2.947 (15 degrees of freedom)
p ≤ 0.01 ≙ t = 3.355 (8 degrees of freedom)

Multiple t-test based on DUNNETT New tables for multiple comparisons with a control: Body weight / food consumption / haematology / clinical biochemistry /organ weights (absolute and relative) (p ≤ 0.01).
The following limits were used:
p ≤ 0.01 ≙ t = 3.09 (36, 33, 32 and 31 degrees of freedom)
p ≤ 0.01 ≙ t = 3.36 (8 degrees of freedom)
p ≤ 0.01 ≙ t = 3.39 (16 degrees of freedom)

For all numerical values (body weight, food consumption and organ weight data) gen-erated from the start of the mating period onwards homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.

Exact test of R.A. FISHER: Histopathology (p ≤ 0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi 2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05) were employed.
These statistical procedures were used for all data. Significantly different data are indicated in the tables.

Indices:

Reproductive indices

For each group the following reproductive indices were determined:

Gestation Index = (number of litters with live pups / number pregnant) x 100

Fertility Index = (number pregnant / number of females evaluated for fertility) x 100

Offspring viability indices

For each litter and group the following reproductive indices were determined:

Birth Index = (Total number of pups born (live + dead)/ Number of implantation scars) x 100

Live Birth Index = (Number of pups born alive on day 0/1 / Total number born (live + dead)) x 100

Viability Index = (number of pups alive on day 4/ number of pups live on day 0/1) x 100

Pre-implantation loss [%] = (corpora lutea - implantations / corpora lutea ) x 100

Post-implantation loss [%] = (implantations - no. pups born alive / implantations) x 100

Historical control data:

none

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Main study / satellite animals: No test item-related mortality was noted. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg test substance/kg b.w./day. Starting at 100 mg test substance/kg b.w./day increased salivation was noted for a very few male and female animals at 100 mg test substance/kg b.w./day and most animals at 200 mg test substance/kg b.w./day during all periods of the study (pre-mating, mating, post-mating, gestation and/or lactation period, respectively) starting on test day 1. Further, at 200 mg test substance/kg b.w./day laboured breathing was observed for one male and rough fur was noted sev-eral females during the pre-mating, mating, gesta-tion and/or lactation period, respectively.

Functional observations (carried out 1 to 2 hours after administration):
Main study animals: No influence was noted on the parameters of the functional observations at any of the tested dose levels for the males.
Females treated with 200 mg test substance/kg b.w./day revealed salivation and pilo-erection.
In addition, hindlimb grip strength of the females was reduced (statistically significant at p ≤ 0.01) by up to 71 % starting at 40 mg test substance/kg b.w./day, though no dose-response relationship was noted. A slight but not significant reduction was also observed for the males at 100 and 200 mg test substance/kg b.w./day.

BODY WEIGHT AND WEIGHT GAIN
Main study / satellite animals: No influence was noted on the body weight of main study and satellite animals during the entire study after treatment with 40, 100 or 200 mg test substance/kg b.w./day.

FOOD CONSUMPTION/WATER CONSUMPTION
Main study / satellite animals:No test item-related influence was observed on the food consumption of the male and female animals treated with either 40 or 100 mg test substance/kg b.w./day during the pre-mating, ges-tation and/or lactation period, respectively.
Treatment with 200 mg test substance/kg b.w./day resulted in a food intake statistically sig-nificant reduced by 11% in test week 2 (pre-mating period) for the male main study animals and for the female main study animals by 10% in test week 1 (pre-mating period) and by 13% on gestation day 7 (gestation period) compared to the control.
The food intake of the female satellite animals treated with 200 mg test substance/kg b.w./day was statistically significant reduced by 20% in test week 1 compared to the control. No influence was noted on the visual appraisal of the drinking water consumption at any of the tested dose levels.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no effect

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no effect

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
no effect

ORGAN WEIGHTS (PARENTAL ANIMALS)
Main study animals: No test item-related influence was noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Main study animals: Macroscopic inspection at necropsy revealed no test item-related changes in the organs or tissues after treatment with either 40, 100 or 200 mg test substance/kg b.w./day.
Recovery period: Satellite animals: An increased salivation was still noted for 2 of 5 males previously treated with 200 mg test substance/kg b.w./day on test days 42 and 43. No test item related influence was noted at the end of the recovery period on test day 58.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The forestomach revealed a squamous cell hyperplasia with submucosal inflammatory reac-tion and hyperkeratosis/parakeratosis in the stra-tum corneum of the animals treated with 200 mg test substance/kg b.w./day. These lesions were completely reversible within the 16-days re-covery period. Further, a dose dependent increase of macro-phages with vacuolization in the mesenteric lymph nodes was observed in the animals treated with 100 or 200 mg test substance/kg b.w./day. The effect was still noted at the end of the recovery period.

OTHER FINDINGS (PARENTAL ANIMALS)
No test item-related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg /kg b.w./day throughout the study up to day 3 post-partum. Treatment with 200 mg /kg b.w./day resulted in a statistically significant (at p ≤ 0.05) increase of the pre-implantation loss by 20.7% compared to the control (10.5%).
The post-implantation loss was not influenced at any dose level.
No female aborted. No malformed pups were noted at birth.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Pre-implantation loss noted at the high dose of 200 mg/kg b.w./day, p.o.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The test substance was assessed for developmental toxicity according to OECD guidline 422. No test item-related mortality was noted, no effects on the pups were noted. Due to the pre-implantation loss noted at the high dose of 200 mg/kg b.w./day, p.o. the no-observed-effect level (NOEL) for reproductive and developmental toxicity was 100 mg/kg b.w./day, p.o. via gavage.

Executive summary:

In a guideline study (OECD 422) the test substance was administered by daily oral gavage to male and female Wistar rats at dose levels of 0, 40, 100 and 200 mg/kg body weight/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 or 36 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 3 days of lactation (for 41 to 56 days).

No test item-related mortality was noted, no effects on the pups were noted. Due to the pre-implantation loss noted at the high dose of 200 mg/kg b.w./day, p.o. the no-observed-effect level (NOEL) for reproductive and developmental toxicity was 100 mg/kg b.w./day, p.o. via gavage.