Decyldimethylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reliable data from guideline studies on reproduction/developmental toxicity are available for C10-DMA, C12-14-DMA, and C16-DMA. These studies were performed according to OECD TG 421/422. For C18-DMA a study according to OECD TG 422 is ongoing.

In addition, the results are supported by read-across data from several DMAOs. The two-generation fertility study and three studies according to OECD TG 422 did not reveal any effects related to reproductive toxicity. No classification according to Regulation (EC) No. 1272/2008 is warranted for the DMA category members. As key value the most conservative value for the category members is selected.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 APRIL 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 50 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).

Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

GLP compliance:

yes

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further this study also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Hylasco Biotechnology Pvt. Ltd., Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19.2 to 24.3°C and relative humidity between 60 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.6– 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 5 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 26 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 5 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

Daily

Details on study schedule:

Experimental starting date: 30 June 2021
Acclimatization: Start: 30 June 2021
End: 04 July 2021
Pre-treatment period: Start: 05 July 2021
End: 18 July 2021
Treatment: Start: 19 July 2021
End: 15 September 2021
Experiment completion: 30 October 2021
Submission of draft report: 31 October 2021
Study completion: 04 April 2022

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes

Details on study design:

The selected male and female rats were assigned to vehicle control and different treatment groups as shown below:
Group No. Group Colour of
cage card Dose
(mg/kg/day) Concent-ration
(mg/mL) Dose volume
(mL/kg) No. of
Rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle control White 0 0 5 10
10 M
F Rab4141
Rab4151 Rab4150
Rab4160
G2 Low dose Yellow 25 5 5 10
10 M
F Rab4161
Rab4171 Rab4170
Rab4180
G3 Mid dose Green 50 10 5 10
10 M
F Rab4181
Rab4191 Rab4190
Rab4200
G4 Mid-intermediate Pink 100 20 5 10
10 M
F Rab4201
Rab4211 Rab4210
Rab4220
G5 High Red 150 30 5 10
10 M
F Rab4221
Rab4231 Rab4230
Rab4240
Recovery Groups
G1R Vehicle control recovery White 0 0 5 5
5 M
F Rab4241
Rab4246 Rab4245
Rab4250
G5R High dose recovery Red 150 30 5 5
5 M
F Rab4251
Rab4256 Rab4255
Rab4260
M: Male; F: Female; Rab: Prefix code for rat numbers
Note: Females were selected with typical 4-5 days oestrous cycles.
From the respective groups, males and females were mated in a 1: 1 ratio (one male to one female) to get at least 8 pregnant rats at or near term/per group.
However, the recovery groups were kept only for observation of reversibility, persistence or delayed occurrence of systemic toxic effects. These animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study was started from the day of first scheduled kill of dam. Recovery animals were sacrificed after completion of 14 Day recovery period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.

b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LDs 0 and 4 were recorded.

c. The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.

d. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

e. After standardization, the individual pup body weight was measured on LD13.

f. The number of nipples/areolae in male pups was counted on LD 13.

g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.

h. The litters were observed daily to note the number of alive, dead and cannibalized pups.

i. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anesthesia. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.
For apparently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of embryos (by staining the implantation sites) for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, pre-coital interval , ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
For two groups, the comparisons of mean between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Reproductive indices:

11.1 Reproductive Performance Data of Parents
a. Male mating index (%)

Number of males with evidence of mating
= ------------------------------------------------------------------- x 100
Number of males cohabited

b. Male fertility index (%)

Number of males siring a litter/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

Number of females mated
= ------------------------------------------ x 100
Number of females cohabited

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of mating

e. Mean number of implantations/group

Total number of implantations
= ---------------------------------------
Total number of pregnant animals


f. Post implantation loss (%)

Number of implantations - Number of live pups
= ------------------------------------------------------------------- x 100
Number of implantations



11.2 Litter Data
a. Mean litter size per group

Total Number of pups born
= -------------------------------------------------
Total Number of littered animals

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

c. Live birth index (%)

No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)


d. Day 4 survival index (%)

Number of viable pups on lactation Day 4
= -------------------------------------------------------- x 100
Number of viable pups born

e. Sex Ratio/ Percentage of male offspring (%)

No. of male pups born
= -------------------------------- x 100
Total

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
There was no mortality observed at any of the doses tested. There were no abnormalities observed in pups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

gross pathology

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect found up to the highest dose.

Key result

Reproductive effects observed:

no

Conclusions:

The results of the study indicated that the oral administration of test item GENAMIN 10 R 3202 D for 2 weeks prior to mating, during mating, and post mating did not cause any toxicological effect on general health, body weights and food consumption in males at 25, 50, 100 and 150 mg/kg/day and at all the tested doses in females. There were no mortalities observed during the treatment at all the tested doses.
At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.
There were no significant changes in neurological parameters, maternal body weights and food consumption during gestation and lactation, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean litter size. No external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
The test item administration did not reveal any treatment related changes in the hematology, coagulation, clinical chemistry, urinalysis parameters, terminal fasting body weights and gross pathology endpoints in the parental rats of either sex. There were no developmental and non-developmental gross findings noted in male or female pups.
Microscopically, hepatocyte hypertrophy was noted in ≥100 mg/kg/day males and was accompanied by higher liver weight at all the doses (non-dose related). The liver findings showed complete reversal at the end of 14-day recovery phase.
There were no test item-related histopathological changes noted in reproductive tissues in any of the parental male and female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.
No Observed Adverse Effect Level

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

The test item was weighed and suspended in vehicle [Corn oil] and administered at the graduated dose levels of 25, 50, 100 and 150 mg/kg bwt/day for low dose (G2), mid dose (G3), Mid-intermediate dose (G4) and High dose(G5)/high dose recovery (G5R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G22702 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2^nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 51 and towards the end of recovery period (Day 64) for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each main group at the end of two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 51), parental females (LD 14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females.

 

Under the experimental conditions employed, the following results were obtained:

Clinical signs and Mortality: There were no clinical signs observed at 25, 50 and 100 mg/kg/day. At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There was no mortality observed at any of the doses tested.

The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

There were no mortalities observed during the treatment at all the tested doses. There were no abnormalities observed in pups.

Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.

 

Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.

 

Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.

 

Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

 

Litter parameters: There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

 

Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes.

 

Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

 

Terminal fasting body weights, organ weights and its ratios: There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related.  The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

 

Gross and histopathology:

There were no test item-related gross lesions observed in both the sexes. There were no developmental and non-developmental gross findings noted in male or female pups.

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes. 

There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.

Reproductive tissues from non-pregnant rats did not show any microscopic changes.

There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

 

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

> 32 mg/kg bw/day (nominal)

Based on:

other: Read-across from C12 DMAO; value converted based on MW

Sex:

male/female

Remarks on result:

other: Original data for the source: >40 mg/kg bw/d

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 32 mg/kg bw/day (nominal)

Based on:

other: Read-across from C12 DMAO; value converted based on MW

Sex:

male/female

Remarks on result:

other: Original data for the source: >40 mg/kg bw/d

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 32 mg/kg bw/day (nominal)

Based on:

other: Read-across from C12 DMAO; value converted based on MW

Sex:

male/female

Remarks on result:

other: Original data for the source: >40 mg/kg bw/d

Reproductive effects observed:

not specified

Executive summary:

The study used as source investigated reproductive toxicity in rats. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From NOV 1979 to NOV 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Two-generation study in rats

GLP compliance:

no

Remarks:

This study was conducted prior to the adoption of GLP compliance standards. However, it was conducted in accordance with the Japanese Ministry of Health and Welfare Guidelines, and was found to be acceptable by the laboratory´s Quality Assurance Unit.

Limit test:

no

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: feed

Vehicle:

not specified

Details on mating procedure:

no data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

approximately 4 months

Frequency of treatment:

daily

Details on study schedule:

no data

Remarks:

Doses / Concentrations:
0, 750, 1500, 3000 ppm for 6.5 weeks, doses were reduced to 0, 188, 375 and 750 ppm. 750 ppm corresponds to 40 mg /kg / day, 375 ppm corresponds to 20 mg/ kg / day, 188 ppm corresponds to 11 mg/ kg / day
Basis:
nominal in diet

No. of animals per sex per dose:

15 males, 30 females

Control animals:

yes, plain diet

Details on study design:

- Dose calculation according to SIDS.

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no data

BODY WEIGHT: Yes
- Time schedule for examinations:
F0:males: weekly, females: weekly until mating, then weighed an days 1, 3, 7, 14, 21 post coitum and on days 1, 4, 11, 18 and 25 post partum
F1: days 1, 4, 11, 18, 25 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: no
- Organs examined: adrenals, all tumors, brain, bone marrow, cecum, duodenum, epididymides, eye and optic nerve, heart, ileum, kidneys, liver, lungs, lymph nodes, mammary glands, esophagus, ovaries, pancreas, pituitary, prostate, seminal vesicles, spleen, stomach, testes, thymus, thyroid, urinary bladder and uterus

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Statistics:

Multiple t-test; Mann-Whitney U-test; x2-test; or Fisher´s Exact Probability test.

Reproductive indices:

no data

Offspring viability indices:

Details for first generation (F1) and second generation (F2)given.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight reductions in weight gain of both parents and offspring, but was without adverse effect on their mating performance and fertility

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight reductions in weight gain of both parents and offspring, but was without adverse effect on their mating performance and fertility

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

> 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slight reduction in weight gain of both parents. Reductions in weight gain did not exceed 10%. The general condition of the animals throughout the study was unaffected by treatment.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a slight reduction in the number of F2 offspring born at the 750 ppm level.

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mating performance, fertility, and conception rate were not affected by treatment in either generation. Gestation and parturition proceeded normally.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the test the NOAEL for the parent generation was established as >40 mg/kg bw/day. NOAEL values for F1 and F2 offsprings were >40 mg/kg bw/day as well. Slight reduction in weight gain of both parents was observed. Reductions in weight gain did not exceed 10%. The general condition of the animals throughout the study was unaffected by the treatment. Mating performance, fertility, and conception rate were not affected by treatment in either generation. Gestation and parturition proceeded normally. There was a slight reduction in the number of F2 offsprings born at the 750 ppm level, however there were no adverse effects of treatment on litter size at birth, live birth index and birth weight in either generation. Therefore, this was not considered an adverse effect.

Executive summary:

15 Male and 30 female Charles River CD rats per dose group were exposed to feed containing 0, 750, 1500, 3000 ppm test substance for 6.5 weeks, doses then were reduced to 0, 188, 375 and 750 ppm. This corresonds to: 0, 11, 20 and 40 mg/kg / day ad libitum for 101 days before mating, throughout mating, gestation and lactation.

Slight reduction in weight gain of both parents was observed. Reductions in weight gain did not exceed 10%. The general condition of the animals throughout the study was unaffected by the treatment. Mating performance, fertility, and conception rate were not affected by treatment in either generation. Gestation and parturition proceeded normally. There was a slight reduction in the number of F2 offsprings born at the 750 ppm level, however there were no adverse effects of treatment on litter size at birth, live birth index and birth weight in either generation. Therefore, this was not considered an adverse effect.

Under the conditions of the test the NOAEL for the parent generation was established as >40 mg/kg bw/day. NOAEL values for F1 and F2 offsprings were >40 mg/kg bw/day as well.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test according to OECD guideline 422 are available for C10-DMA, C12-14 DMA, and for C16-DMA. For C18-DMA guideline a study according to OECDT TG 422 is ongoing. For C12-14-DMA a supporting reproduction/developmental toxicity screening test (OECD guideline 421) is also available.

In the two newly available gavage studies performed in Wistar rats according to OECD TG 422 with C10-DMA, and C12-14 DMA the NOAEL for both, parental and F1 animals was 150 mg/kg bw/d, the highest dose tested. The same NOAEL was derived in the third OECD TG 422 with C16-DMA for females, while the NOAEL for males was 100 mg/(kg bw x d) based on reduced body weight and weight gain (no effects on reproductive parameters observed). No further treatment related adverse effects on systemic, reproduction and fertility parameters up to and including the highest dose could be observed in any of these studies.

In the supporting reproduction/developmental toxicity screening test (OECD guideline 421) for C12-14-DMA, Sprague-Dawley rats were dosed with 0, 50, 150, 300 and 450 mg/kg bw/d by gavage. The NOEL for both parent animals and their pups was 50 mg/kg bw/d. 150 mg/kg bw/d were in the beginning lethal range for pregnant rats and in the lethal range for embryos/fetuses/pups. No effects were observed on estrous cycle and reproductive performances. However, at 150 mg/kg bw/d parturition of two dams was influenced. In addition, as outlined in the section on repeated dose toxicity, at 150 mg kg bw/d lethality was already reported (2/10 females died). The NOEL for fertility is 50 mg/kg bw/d.

Overall, in all available studies, no indication of any effect on fertility, reproduction, gestation, partition, growth of offspring or lactation was found beyond the dose levels of apparent maternal toxicity.

Based on these observations, the DMAs of this category do not have to be classified for effects on fertility according to Regulation (EC) No 1272/2008.

Effects on developmental toxicity

Description of key information

Reliable data from guideline studies on reproduction/developmental toxicity are available for C10-DMA, C12-14-DMA and C16-DMA. These studies were performed according to OECD TG  422. No indication of developmental toxicity was found. For C18-DMA a study according to OECD TG 422 is ongoing. And either for C12-14 DMA or C16-DMA a OECD TG 414 is planned. In addition, the unlikeliness of developmental toxicity for DMA is supported by read-across data from several DMAOs. A developmental toxicity study with C12-14 DMAO is currently used to cover the information requirements for this endpoint. The three new studies according to OECD TG 422 did not reveal any effects related do developmental toxicity. No classification according to Regulation (EC) No. 1272/2008 is warranted for the DMA category members.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Species:

rabbit

Dose descriptor:

NOAEL

Effect level:

> 129 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from C12 DMAO; value converted based on MW

Remarks on result:

other: Original data for the source: >160 mg/kg bw/d

Dose descriptor:

NOAEL

Effect level:

> 129 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from C12 DMAO; value converted based on MW

Remarks on result:

other: Original data for the source: 160 mg/kg bw/d

Abnormalities:

not specified

Developmental effects observed:

not specified

Executive summary:

The study used as source investigated developmental toxicity in rabbits. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From MAY 1980 to OCT 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Test for developmental toxicity.

GLP compliance:

no

Remarks:

This study was conducted prior to adoption of GLP compliance standards. However, it was conducted in accordance with the Japanese Ministry of Health and Welfare Guidelines, and was found to be acceptable by the laboratory´s Quality Assurance Unit

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

no data

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Animals were artificially inseminated using pooled semen from New Zealand White bucks.

Duration of treatment / exposure:

days 6-18 (inclusive) of gestation

Frequency of treatment:

daily

Duration of test:

approximately 5 months

No. of animals per sex per dose:

14 female animals per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

no data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily, reported on days 0, 6, 8, 10, 12, 14, 16, 18, 23, 28.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: no data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: Yes: (all)
- Head examinations: No

Statistics:

Multiple ´t´-test, ´t´-test, Mann-Whitney U-test, Chi-squared test and Fisher´s Exact Probability test (Armitage modification).

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: details see below

Details on maternal toxic effects:
Maternal condition was generally unaffected by treatment with the test material; however, maternal bodyweight gain was depressed in all treated groups, although at 40 mg/kg/day terminal bodyweights similar to those of controls were achieved. Three females receiving 80 mg/kg/day and three females receiving 160 mg/kg/day died or were killed in extremis, but no direct involvement of the test substance was apparent. Food intake, when compared with pre-treatment values, was reduced during the second half of the treatment period in groups receiving 40 and 80 mg/kg/day, and from the commencement of treatment in animals receiving 160 mg/kg/day. Water intake was also decreased in all treated groups. Reductions in body weight gain, food intake and water intake did not exceed 10%.

Dose descriptor:

NOAEL

Effect level:

> 160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter response was unaffected by any treatment with the test material. No adverse effects upon litter responses and development were recorded. No teratogenic responses were observed.

Dose descriptor:

NOAEL

Effect level:

> 160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of the test, the maternal animals were generally unaffected by treatment with the test material; however, maternal bodyweight gain was depressed in all treated groups, although at 40 mg/kg/day terminal bodyweights similar to those of controls were achieved. Food intake, when compared with pre-treatment values, was reduced during the second half of the treatment period in groups receiving 40 and 80 mg/kg/day, and from the commencement of treatment in animals receiving 160 mg/kg/day. Water intake was also decreased in all treated groups. Reductions in body weight gain, food intake and water intake did not exceed 10%. The NOAEL for maternal toxicity was established as > 160 mg/kg bw/day
Litter response was unaffected by any treatment with the test material. No adverse effects upon litter responses and development were recorded. No teratogenic responses were observed. The NOAEL for teratogenicity was established as > 160 mg/kg bw/day

Executive summary:

14 New Zealand white rabbits per dose group were exposed to 0, 40, 80 or 160 mg test substance/kg bw/day during days 6 -18 og gestation.

Under the conditions of the test, the maternal animals were generally unaffected by treatment with the test material; however, maternal bodyweight gain was depressed in all treated groups, although at 40 mg/kg/day terminal bodyweights similar to those of controls were achieved. Food intake, when compared with pre-treatment values, was reduced during the second half of the treatment period in groups receiving 40 and 80 mg/kg/day, and from the commencement of treatment in animals receiving 160 mg/kg/day. Water intake was also decreased in all treated groups. Reductions in body weight gain, food intake and water intake did not exceed 10%.

Litter response was unaffected by any treatment with the test material. No adverse effects upon litter responses and development were recorded. No teratogenic responses were observed.

The NOAEL for maternal toxicity was established to be > 160 mg/kg bw/day.

The NOAEL for teratogenicity is > 160 mg/kg bw/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

N/A

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (EPA OTS) and according to GLP.

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: Females: 65 days; Males: 42 days.
- Weight at study initiation: Females: 218 - 262 g; Males: 506 - 969 g.
- Housing: Rats were individual housed except during cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat's cage.
- Diet (ad libitum): Certified Rodent Diet #5002 (Purina Nutrition International, St. Louis, Missouri) in individual feeders.
- Water (ad libitum): Local water that had been processed by passage through a reverse osmossis membrane (R.O. water) was available to the rats from an automatic watering system and/or individual water bottles. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): 30 to 70
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: N/A To: N/A

Route of administration:

oral: gavage

Vehicle:

other: Sterile Water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substances were prepared weekly at the Testing Facility. Dosage calculations were adjusted for the 32% (w/v) concentration of the test substance. Prepared formulations were stored at room temperature and stirred continuously (magnetic stir plate with stir bar) during dosage administration.


DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Storage temperature of food: Room temperature.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Sterile Water
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- Proof of pregnancy: sperm in vaginal smear and/or a copulatory plug observed in situ will be referred to as day 0 of pregnancy

Duration of treatment / exposure:

Days 6 through 19 of presumed gestation.

Frequency of treatment:

Daily

Duration of test:

Approximately 4 weeks.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of a dosage-range study (Angus Research Laboratories, Inc., Protocol 916-025P), in which dosage levels of 0, 32.5, 100, 325 and 650 mg/kg/day were evaluated.
Dosages of 325 and 650 mg/kg/day were excessively toxic to the dams and conceptuses. At the 32.5 and 100 mg/kg/day dosage levels, no developmental toxicity was observed. Excess salivation was observed at the 100 mg/kg/day dosage level, and slight, non-statistically significant decreases in maternal body weight gains and feed consumption values were observed at the 32.5 and 100 mg/kg/day dosage levels. Doses of 200, 100, and 25 mg akly dimethyl amine oxide/kg/day were chosen.
- Rationale for animal assignment (if not random): Unpon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Female rats were assigned to four dosage groups (Groups I through IV), twentry-five rats per dosage group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations checked were: mortality, moribundity, pertinent behavioral changes and other signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly and Rats were also examined for clinical observations immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimation, on DG 0, daily during the dosage period and on DG 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

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
- Other: A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses are considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions are differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetuses was a late resorption.

Fetal examinations:

- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Bionomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and feal anomaly data) were analyzed using Bartletts' Test of Homogeneity of Variances and the Analysis of Variance, when appropriated [i.e., Bartlett's Test was not significant (pCount data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: details see below

Details on maternal toxic effects:
MORTALITY:
Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.
A single rat in the 200 mg/kg/day dosage group was found dead on day 19 of gestation (DG 19) after 13 daily dosages. This dam lost body weight after DG 9 and had reduced feed consumption values throughout the study. Adverse clinical observations included excessive salivation (DGs 8 to 9, 14 and 17 to 18), gasping (DGs 9 to 11 and 17 to 18), labored breathing (DGs 10 to 13 and 16 to 18), ungroomed coat (DGs 10 to 13), brown perianal substances (DGs 12 to 13), urine-stained abdominal fur (DGs 12 to 14 and 16 to 18), brown perivaginal substance (DGs 12 to 15), chromorhinorrhea (DG 15), rales (DG 16) and emaciation (DGs 17 to 18). No gross lesions were revealed by necropsy; all tissues appeared normal for moderate degree autolysis. There were 17 early resorptions in utera. This death was attributed to effects of the test substance because similar observations occurred in surviving rats in this dosage group.
A single rat in the 200 mg/kg/day dosage group was found dead on DG 18 after 12 daily dosages. This death was attributed to an intubation accident. This dam lost body weight after DG 13 and had reduced feed consumption values throughout the study. Adverse clinical observations included rales (DGs 10 to 11 and 13), excessive salivation (DGs 13 to 14), gasping (DGs 13 to 14), labored breathing (DGs 13 to 17), red perioral substance (DGs 15 to 17), urine-stained abdominal fur (DGs 15 to 17), dehydration (DG 16) and emaciation (DG 17). External observations at necropsy included red substance on fur of the nose, forepaws and forelimbs. Gross necropsy revealed a tear in the esophagus; all other tissues appeared normal for slight degree of autolysis. There were 14 fetuses in utero. The viability of the fetuses could not be determined because of the maternal death.

CLINICAL OBSERVATIONS:
Adverse clinical observations occurred at increased incidences in the 100 and 200 mg/kg/day dosage group rats. Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased (pAll other clincal observations were considered unrelated to the test substance because: 1) the incidences were not dosage-dependent; and/or 2) the observations occurred in only one or two rats. These clinical observations included one 200 mg/kg/day dosage group dam with an axillary mass attributed to an intubation error, and localized alopecia (limbs and underside) in one or two rats in the 0 (Vehicle), 25, and 200 mg/kg/day dosage groups.

NECROPSY OBSERVATIONS:
All necropsy observations were considered unrelated to the test substance because they were single events. These observations included slight dilation of the pelvis of the right kidney of one vehicle group dam and one 200 mg/kg/day dosage group dam. A single dame also had a distended urinary bladder with ten calculi and thickened and red bladder walls. One 100 mg/kg/day dosage group dam had large adrenals, four dark red areas on the fundic mucosa and numerous raised tan areas on the pyloric mucosa of the stomach, the intestines and stomach were distended with gas and the left lateral lobe of the liver was mottled. One 200 mg/kg/day dosage dam had a mass in the left axilla in-life; at necropsy, a clear tan gelatinous fluid was located subcutaneously in the area of the ventral neck, left axilla and leateral chest, the esophagus had a thickened area and the plyoric folds of the stomach were thickened. These observation were presumed to be the sequelae of a presumed intubation error.

MATERNAL BODY WEIGHTS, GRAVID UTERINE WEIGHTS AND BODY WEIGHT CHANGES:
Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced (pThe gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced (pBody weights and body weights gains were unaffected at dosages of 25 mg/kg/day of the test substance. Gravid uterine weights were unaffected by the 25 and 100 mg/kg/day dosages of the test substance.

MATERNAL ABSOLUTE (g/day) and RELATIVE (g/kg/day) FEED CONSUMPTION VALUES:
Absolute (g/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced (p
CAESAREAN-SECTIONING:
Caesarean-sectioning observations were based on 24, 25, 24 and 22 pregnant rats in the 0 (vehicle), 25, 100 and 200 mg/kg/dosage groups, respectively. Male and female fetal body weights were significantly reduced (pNo other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 200 mg/kg/day. The litter averages for corpora lutea, implantations, late resorptions, percent resorbed conceptuses (calculated excluding the completely resorbed litter in the 200 mg/kg/day dosage group), and percent live male fetuses were comparable among the four dosage groups and did not significantly differ. There were no dead fetuses.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: details see below

Details on embryotoxic / teratogenic effects:
FETAL ALTERATIONS:
Fetal alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); or 2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.
Fetal evaluations were based on 339, 375, 348, and 297 Ceasarean-delivered live fetuses in 24, 25, 24, and 21 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. Each fetus was examined for gross external alterations, approximately one half of the fetuses in each litter were examined for soft tissue alterations and the remaining fetuses were examined for skeletal alterations and the number of ossification sites.
The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased (pAll other gross external, soft tissues or skeletal alterations (malformations or variations) were considered unrelated to the test substance because 1) the litter and fetal incidences were not dosage-dependent; 2) the alteration occurred in only one fetus; or 3) the incidences were within the ranges observed historically at the Test Facility.

FETAL ALTERATIONS:
In groups I through IV, litters with fetuses with alterations numbered 8 (33.3%), 11 (44.0%), 8 (33.3%), and 15 (71.4%), respectively. The numbers of fetuses with any alterations were 11 (3.2%), 25 (6.7%), 14 (4.0%) and 32 (10.8%) respectively. The percentages of fetuses with any alteration per litter were 3.6%, 6.4%, 4.5% and 11.5% in the four respective dosage groups.
The significant increases in the percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were considered to reflect delays in skeletal ossification, related to the significantly reduced (p
FETAL GROSS EXTERNAL ALTERATIONS:
One vehicle group fetus had whole body edema (anasarca); this fetus also had an absent innominate artery at soft tissue examination. One 25 mg/kg/day fetus had a thread-like tail; at skeletal evaluation, this fetus had fused arches of the 3rd sacral vertebra and no caudal vertebrae. One 200 mg/kg/day group fetus had a kinked tail, the 4th and 5th digits of the lift hindlimb were fused and a skin tab located to the left of its tail. At skeletal evaluation of this fetus, further evaluation of the skin tab revealed two bones (possibly a femur and fibula) fused together on the left side of the pelvis. An extra paw appeared to be attached to the underside of the left paw; also the centra of the 6th and 11th thoracic vertebrae were bifid. One 200 mg/kg/day fetus had a micrognathia; soft tissue evaluation of this fetus revealed a small tongue.

MALFORMATIONS - SOFT TISSUE EVALUATION:
One 200 mg/kg/day fetus had a small tongue; micrognathia was identified at gross external evaluation.

VARIATIONS - SOFT TISSUE EVALUATION:
One control group fetus and one 25 mg/kg/day dosage group fetus had an absent innominate vessel; whole body edema (anasarca) was identified at gross external evaluation for the control fetus, also an additional fetus had the umbilical artery descending to the left of the urinary bladder.

MALFORMATION - SKELETAL ALTERATIONS:
One control group fetus had fused arches of the 4th cervical vertebra; additional variations in ossification occurred in this fetus (unossified 1st sternal centra, incompletely ossified pubes).
One 25 mg/kg/day dosage group fetus had fused arches of the 3rd sacral vertebra; this fetus also had no caudal vertebrae (thread-like tail was noted at gross external examination).

VARIATIONS - SKELETAL ALTERATIONS:
A cervical rib was present at the 7th vertebra in one 25 mg/kg/day fetus. This fetus had no other skeletal alterations.
A bifid centrum in the thoracic vertebrae occurred in 1, 6, 5 and 9 fetuses from 1, 3, 5 and 8 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups respectively. One fetus in the 25 mg/kg/day dosage group also had incompletely ossified 1st and 2nd sternal centra, a fetus in the 100 mg/kg/day dosage group also had a bifid centrum of the 1st lumbar vertebra and a fetus in the 200 mg/kg/day dosage group also had incompletely ossified pubes.
An incompletely ossified arch in the 6th lumbar vetrebra occurred in one 100 mg/kg/day dosage group fetus; this fetus also had incompletely ossified ischia and pubes.
The significant increase (pDelayed sternal ossification (incompletely ossified and/or not ossified 1st and/or 2nd sternebrae) occurred in 4, 8, 3 and 11 fetuses from 4, 6, 2 and 7 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidence of only incompletely ossified 1st sternal centra was also significantly increased (pThe significant increases (pThe pubes and/or ischia were incompletely ossified in 8, 12, 7 and 16 fetuses from 6, 4, 3 and 6 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidenc of only incompletely ossified pubes was significantly increased (pThe significant increase (pThe litter averages for ossified caudal vertebrae, sternal centers and metacarpals per fetus were significantly decreased (pAnalyses of the average numbers of fetal ossification sites per fetus did not reveal any other statistically significant differences among the four dosage groups. Ossification of the hyoid, vertebrae (cervical, thoracic, lumbar, and sacral) ribs, sternum (manubrium and xiphoid), forelimbs (carpals and phalanges) and hindlimbs (tarsals, metatarsals and phalanges) occurred at similar incidences in litter in all dosage groups.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: delays in skeletal ossification

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 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

          

Conclusions:

On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) for the test substance (alkyl dimethyl amine oxide) is 25 mg/kg/day. The 200 mg/kg/day dosage caused mortality in one dam, the 100 and 200 mg/kg/day dosages caused adverse clinical observations, reductions in body weight gain and reduced feed consumption values. Reductions in relative feed consumption values were also noted in the 25 mg/kg/day dose group; however, no concomitant adverse effects were noted at this dosage level. The developmental NOAEL was 25 mg/kg/day; the 200 mg/kg/day dosage caused reduced fetal body weights and associated delays in skeletal ossification and the 100 mg/kg/day dosage also caused delays in skeletal ossification.

Executive summary:

One-hundred Crl:CDBR VAF/Plus presumed pregnant female rats were randomly assigned to four dosage groups (Groups 1 through IV), 25 rats per group. The test substance preparations for dosing were corrected for purity. The test substance, was administered orally (via gavage) once daily to these female rats on days 6 through 19 of presumed gestation (DGs 6 through 19), at dosages of 0 (Vehicle), 25, 100, and 200 mg alkyl dimethyl amine oxide/kg/day. The dosage volume was 5 mL/kg, adjusted daily on the basis of the individual body weights recorded before intubation. The rats were intubated at approximately the same time each day.

The rats were observed for viability at least twice a day and for general appearance weekly during acclimation and on DG 0. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20). Body weights were recorded weekly during acclimation, on DG 0, daily during the dosage period and on DG 20. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18, and 20.

All surviving rats were sacrificed on DG 20, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group in order to minimize bias. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses and early and late resorptions. The gravid uterus was weighed. Each fetus was removed from the uterus and subsequently weighed and examined for sex and gross external alterations. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection techniques. The remaining fetuses in each litter were examined for skeletal alterations.

Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.

Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased numbers of 200 mg/kg/day dosage group rats. Additionally, chromorhinorrhea occurred in one and two rats in these two dosage groups, respectively. Observations of brown or red perivaginal substance, emaciation, brown perianal or perinasal substance, dehydration, ungroomed coat and soft or liquid feces occurred in one or two rats in the 200 mg/kg/day dosage group. All necropsy observations were considered unrelated to the test substance.

Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced body weight gains for the entire dosage period (calculated as days 6 to 20 of gestation) and body weight gains were significantly reduced in the 200 mg/kg/day dosage group for the entire gestation period (DGs 0 to 20). Body weights were significantly reduced in the 200 mg/kg/day dosage group on DGs 11 through 20. The gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced in the 200 mg/kg/day dosage group.

Absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced in the 200 mg/kg/day dosage group. Relative feed consumption values for the entire dosage period were also significantly reduced in the 100 mg/kg/day dosage group, while values for the gestation period were significantly reduced in the 25, 100 and 200 mg/kg/day dosage groups.

Male and female fetal body weights were significantly reduced in the 200 mg/kg/day dosage group. Live litter size was decreased and the number of early resorptions was increased in the 200 mg/kg/day dosage group, but apparently as the result of one dam in this dosage group that had a litter consisting of 16 early resorptions.

The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced fetal body weights in this group. These delays in ossification included significant increases in the fetal and/or litter incidences of bifid thoracic vertebrae centra, incompletely and/or not ossified 1st or 2nd sternal centra, incompletely ossified pubes and significant decreases in the numbers of ossified caudal vertebrae, sternal centers and metacarpals. Additionally, delays in ossification occurred in the 100 mg/kg/day dosage group and included a significant increase in the litter incidence of bifid thoracic vertebrae centra.

NOAEL for both maternal toxicity and developmental toxicity is 25 mg/kg/day.

Reference 4

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Species:

rat

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

other: Read-across from C12-14 DMAO; value converted based on MW

Remarks on result:

other: Original data for the source: 25 mg/kg bw/d

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

other: Read-across from C12-14 DMAO; value converted based on MW

Basis for effect level:

other: Original data for the source: 25 mg/kg bw/d

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

79 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

Overall developmental toxicity: Value reported above is based on read-across from C12-14 DMAO, the value is converted based on molecular weight (value for the source substance: 100 mg/kg bw/d).

Executive summary:

The study used as source investigated developmental toxicity in rats. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good

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

For C10-DMA, C12-14 DMA and C16-DMA reliable combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test according to OECD guideline 422 are available. For for C18-DMA an OECD TG 422 is ongoing. Either for C12-14 DMA or C16-DMA an OECD TG 414 is planned. For C12-14-DMA a supporting reproduction/developmental toxicity screening test (OECD guideline 421) is also available.

Currently the information requirements for this endpoint can be fulfilled by read across from DMAOs, for which a qualified and reliable OECD Guideline 422 study (C12-18-DMAO) and a reliable developmental toxicity study according to EPA OTS 798.4900 (C12-14-DMAO) are available.

In addition, two developmental toxicity studies (one in rats for C10-16-DMAO, and one in rabbits for C10-16-DMAO) as well as a two- and a three-generation study for DMAO, are reported.

In the reproduction/developmental toxicity screening test (OECD guideline 422) for C10-DMA, C12-14 DMA and C16-DMA Wistar rats were exposed via gavage to 15, 50, 100 or 150 mg/kg bw/d. No treatment-related effects on implantation and mean litter size were observed. In addition, no external abnormalities in live or dead pups, no treatment-related changes in the ano-genital distance, ano-genital ratio or pup body weights were observed in any of the dose groups. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested. Therefore, the NOAEL for developmental toxicity in these studies is ≥ 150 mg/kg bw/d, the highest dose tested.

In the reproduction/developmental toxicity screening test (OECD guideline 421) for C12-14-DMA, rats were dosed with 0, 50, 150, 300 and 450 mg/kg bw/d by gavage. The NOEL for both parent animals and their pups was 50 mg/kg bw/d. 150 mg/kg bw/d were in the beginning lethal range for pregnant rats and in the toxic range for embryos/fetuses/pups. At 150 mg/kg bw/d, the number of pups alive was reduced. However, effects on pups were observed in a dose range where maternal toxicity was already evident.. Regarding developmental toxicity the effects on the pubs were clearly secondary to maternal toxicity in the OECD 421 study.

In the reliable developmental toxicity study with C12-14-DMAO (reliability 1, according to EPA OTS 798.4900) a lower NOAEL of 25 mg/kg bw/d for maternal and developmental toxicity based on adverse clinical observations, reduction in body weight gain and reduced feed consumption in dams as well as reduced fetal body weights and associated delays in skeletal ossification in the higher dose groups (100 and 200 mg/kg bw/d) in pups is reported.

In the supporting study with C12-18-DMAO (OECD TG 422), a developmental NOEL of 100 mg/kg bw/d due to pre-implantation loss in the higher dose group (200 mg/kg bw/d) was determined. No other effects on development of pups were observed. The NOEL for maternal toxicity was 40 mg/kg bw/d, due to local and systemic toxicity at 100 mg/kg bw/d or higher doses.

Two additional studies on DMAOs support the data presented so far with NOAEL values for developmental toxicity of 100 mg/kg bw/d (rats, Lion Corporation, 1979B) and >160 mg/kg bw/d (rabbits, Lion Corporation, 1980):

In the study by Lion Corporation, (1979B), rats were exposed to 0, 50, 100 or 200 mg C10-16-DMAO/kg bw/d from day 7 to 17 of gestation. In pups from females killed on day 20 of gestation, mean fetal weight was depressed at the highest dose level, and this was associated with a slight retardation of fetal ossification. No other effects were observed. The NOAEL for maternal toxicity and teratogenicity was 100 mg/kg bw/d.

In a study on developmental toxicity in rabbits (Lion Corporation, 1980), animals were exposed to 0, 40, 80 or 160 mg C10-16-DMAO/kg bw/d during days 6-18 of gestation. Litter response was unaffected by any treatment with the test material. No adverse effects upon litter responses and development were recorded. No teratogenic effects and no maternal toxicity were observed. The NOAEL for maternal toxicity and teratogenicity in this study was 160 mg/kg bw/d.

The results of the two- and the three-generation studies support the findings reported above. In the two-generation study, rats were exposed to 0, 11, 20 and 40 mg C10-16-DMAO/kg bw/d. The NOAEL values for the parent, the F1 and the F2 generation were 40 mg/kg bw/d. In the three-generation study, rats were exposed to 0, 0.005, 0.02 and 0.1% C10-16-DMAO in diet. No adverse effects were observed at any dose level, resulting in a NOEL of 0.1%, the highest concentration tested (40 mg/kg bw/d for males and 50 mg/kg bw/d for females).

In all available studies no indication of developmental toxicity was found beyond the dose levels of apparent maternal toxicity.

Based on these observations, the DMAs of this category do not have to be classified for developmental effects according to Regulation (EC) No 1272/2008.

Justification for classification or non-classification

Based on the observation that effects on fertility and developmental toxicity were found beyond the dose levels of apparent maternal toxicity, the DMAs of this category do not have to be classified for reproductive toxicity and effects on or via lactation according to Regulation (EC) No 1272/2008.