Reaction mass of bis(N-decyl-N,N-dimethyldecan-1-aminium) carbonate and N-decyl-N,N-dimethyldecan-1-aminium hydrogen carbonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Reliability: GLP study according to EU- and/or OECD-guidelines - Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 204.2-205.8 g; Females: 153.3-155.2 g
- Housing: stainless steel, wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ca. 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): constant
- Humidity (%): constant
- Photoperiod (hrs dark / hrs light): 12 hrs

IN-LIFE DATES:
- From: 31 March 1988
- To: 15 August 1989

Route of administration:

oral: feed

Vehicle:

ethanol

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): standard rodent chow
- Storage temperature of food: room temperature

Details on mating procedure:

Twenty-eight F0 rats/sex/dose were bred twice to produce F1A and F1B litters. Selected males and females from F1B litters were used as parents of the F2 generation. Twenty-eight (28) F1 pups/sex/dose, exposed to the same dietary concentration of test item as their parents, were subsequently bred twice to produce F2A and F2B litters. Details on the procedures followed are provided in the following narrative.
- Pre-Breed Exposures: All initial animals (F0 generation) were weighed during quarantine just prior to the start of exposures and 112 per sex were randomly distributed, stratified by body weight, into four dose groups using a computer program. Twenty-eight (28) animals per sex were assigned to each dose group. Animals were not placed on study if they exhibited clinical signs during quarantine, or if their body weight exceeded +/- 20% of the mean weight for each sex. At the start of test chemical administration the mean weight for males for all four groups ranged from 204.2 - 205.8 g; for females, mean weight ranged from 153.3 - 155.2 g. The approximate age of F0 study animals was six (6) weeks. Animals received in the initial shipment and not used in the study were euthanized and discarded. The study animals, housed singly, were exposed to test item in the diet, ad libitum, at 0, 300, 750 or 1500 ppm for ten (10) consecutive weeks. During this pre-breed treatment period, the animals were examined twice daily for mortality and moribund status and once daily for any clinical signs of toxicity. They were weighed weekly and food consumption also was measured weekly. These measurements allowed for calculation of the amount of chemical consumed, expressed as mg test article/kg of body weight.
- Mating: After the 10-week pre-breeding exposure period was completed, the animals within each dose group were randomly mated, one male to one female, to produce the F1A generation. The following mating procedure was used: after the first seven days of the mating period, females of unsuccessfully mated pairs were placed with males of other unmated pairs within the same dose group; after an additional seven days, unsuccessfully mated pairs were switched again for a period of seven days or until successful mating had occurred, whichever came first, allowing for a period of 21 days to mate. The observation of a dropped copulation plug or the presence of vaginal sperm was considered evidence of successful mating. The area under the females' cage was examined for dropped copulation plugs twice daily (a.m. and p.m.) during the cohabitation period. Vaginal smears were obtained once daily (a.m.). The day a copulation plug or sperm positive smear was observed was designated gestational day (gd) 0 (Hafez, 1970). Once a plug was observed, the male and female from that mating pair were individually housed. For any mating pairs which did not show evidence of successful mating, the last scheduled mating day was considered gd 0 for that female and the animals were treated accordingly for subsequent events. Mated females were weighed on gd 0, 6, 15 and 20. On gd 20, each female was transferred to a shoe-box cage. Females were observed twice daily beginning on gd 20 for evidence of littering. Dams with litters were weighed on postnatal days 0, 7, 14 and 21. Food consumption was measured in three (3)- to four (4)-day intervals throughout gestation (gd 0-20) and, postnatally, from lactational day 0 through 14. The dams were allowed to rear their young to day 21 postpartum. On day 21 postpartum, each litter was weaned but the weanlings remained housed as litters for an additional seven days. As each F1A litter reached day 28 postpartum, pups from each litter were randomly selected for necropsy on the basis of a computer-generated random selection scheme for a total of ten (10) male and 10 female pups per dose group. The remaining FIA pups were examined grossly and then euthanized and discarded following evaluation of necropsy findings in their selected littermates.
At least ten (10) days after the weaning of the F1A litters, F0 males and females were mated a second time employing previously described mating procedures. When possible, previously nonpregnant females (or males failing to induce pregnancy) were mated to animals which were successful at the first mating. The mating, gestation, lactation, and weaning were performed as described above. As each F1B litter reached day 28 postpartum, pups from each litter were randomly selected as parents to produce the F2 generation; a total of 28 male and 28 female pups per dose group were selected. In addition, ten (10) F1B pups per sex per dose were randomly selected for necropsy. Selections were based on a computer-generated random selection scheme. Remaining F1B pups were examined grossly and then euthanized and discarded. The FO parental females were necropsied after the FIB litters were weaned; FO males were necropsied after parturition of the first FIB litters.
F1B animals selected to be parents of the F2 generation (hereafter referred to as F1 adults) were administered the test diets for ten weeks as described above. The average body weight of F1 males ranged from 260.3-309.0 g at the start of the pre-breed period; the F1 females weighed an average of 171.6-200.8 g. Their approximate age was four to six weeks. The F1 animals were approximately 14 to 16 weeks of age at the initiation of the mating period. They were mated as described above for the F0 animals, including, but not limited to, pairing one male:one female, cohabitation for 21 days or until a copulation plug is observed, whichever comes first, and transfer of pregnant females to appropriate caging on gd 20. Brother-sister matings were avoided whenever possible. The F1 animals were mated twice to produce F2A and F2B litters as described above for the F0 animals.
- Offspring: All pups from the F1A, F1B, F2A and F2B generations were examined as soon as possible on the day of birth to determine the number of viable and stillborn male and female members of each litter. Litters were evaluated twice daily for survival. On day 4 after birth, the size of each litter was adjusted by eliminating extra pups selected randomly by computer to yield, as nearly as possible, four males and four females per litter. Culled pups were examined externally, sacrificed, and discarded. Survival indices were calculated at 0, 4, 7 and 14 days after birth, at weaning on Postnatal Day 21, and one week subsequent to weaning. The sex of each pup was determined and verified daily. All live pups were weighed individually at 0, 4, 7 and 14 days after birth and at weaning on Postnatal Day 21. All pups were also weighed on Postnatal Day 28. The body weights and sexes were recorded on an individual basis but the pups were not uniquely identified until selection on Day 28. All pups were examined for physical abnormalities at birth and throughout the pre-weaning period. All pups dying during lactation were necropsied when possible to investigate the cause of death.
All FIA and FIB littermates were allowed to remain together for a minimum of seven days after weaning. In FIA litters ten (10) pups of each sex per dose group were randomly selected for necropsy. Following evaluation of gross findings and with permission of the Sponsor's Representative, the remaining F1A pups were examined for gross external abnormalities, euthanized and discarded.
For the F1B litters, twenty-eight (28) males and 28 females from each dose group were selected on a random basis to become parents of the next generation (F1 parents to produce F2 litters). All pups were available for selection except those not expected to survive because of physical abnormalities. The parentage of each weanling was ascertained to avoid brother-sister matings if possible. In addition ten (10) FIB pups of each sex from each dose group were also randomly selected for necropsy (see below). Following evaluation of gross findings at necropsy of their littermates, the remaining offspring were examined for gross external abnormalities, euthanized and discarded.
The F2A and F2B litters were evaluated exactly as described above for the F1A and F1B litters except no animals were selected to become parents of the next generation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test item in the test diets was determined using a Hewlett-Packard 5880A gas chromatograph with Nitrogen-Phosphorus detection. For the first four weeks of the study, test diets from all levels were analyzed for concentrations of test item prior to administration of the diets to the animals. For the remaining 60 weeks of the study, all test diets from every fourth preparation were analyzed after administration of the diets to the study animals. Homogeneity and stability analyses of the test diets were performed prior to the start of test diet administration.
The results of the homogeneity study indicated that the distribution of test item in the test diet was uniform. The stability study indicated that the dosed feed was stable at room temperature for 21 days when stored in closed polyethylene containers and for at least 14 days when stored in open glass jars at room temperature. The dose levels selected were 0, 300, 750 and 1500 ppm.
The periodic analyses of the dosed feed indicated that the mean concentrations of test item in the diet for the 300, 750 and 1500 ppm dosage levels were 96.2 - 108.8 % of nominal for 300 ppm, 98.8 - 108.8 % of nominal for 750 ppm and 94.5 - 109.2 % of nominal for 1500 ppm, respectively.
No test chemical was detected in any of the control diets with a minimum detection limit of 5 ppm.

Duration of treatment / exposure:

F0: 27 weeks (from 1st prebreed dose to last F0 sacrifice)
F1: 32 weeks (from 1st F1B wean to last F1B sacrifice)
F2B: to weaning

Frequency of treatment:

Ad libitum
Actual doses were age dependent (the following ranges are approximate)
- Males:
16-32 mg/kg/d at 300 ppm
38-79 mg/kg/d at 750 ppm
79-149 mg/kg/d at 1500 ppm
- Females:
20-34 mg/kg/d at 300 ppm
49-82 mg/kg/d at 750 ppm
99-154 mg/kg/d at 1500 ppm

Details on study schedule:

See details on mating procedure above.

Remarks:

Doses / Concentrations:
0, 300, 750 and 1500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

28/sex

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Three graduated dosage levels of the test material were evaluated in three groups of rats, anticipating that at the higher dosage level(s) some toxicological or pharmacological effect(s) will be observed and that at the lower dosage level(s) no treatment-related effects will be seen.
- Rationale for animal assignment (if not random): random

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once each week.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreed and mating for both sexes. For females on gestational day (gd) 0, 6, 15 and 20 and on postnatal day (pnd) 0, 7, 14 and 21. All pups were weighed on postnatal day (pnd) 1, 4, 7 and 14, and at weaning (day 21).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: Weekly during prebreed for both sexes. For females in 3- or 4-day intervals throughout gestation and to pnd 14.

Oestrous cyclicity (parental animals):

See section on mating procedure above

Sperm parameters (parental animals):

See section on mating procedure above

Litter observations:

See section on mating procedure above

Postmortem examinations (parental animals):

Gross and Microscopic Pathology: All F0 and F1 parental animals in all groups (both generations) were euthanized by severing the brachial blood vessels following anesthesia with methoxyflurane, and subjected to a complete necropsy. The necropsy included: examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. Sacrifice of parental males occurred after the second mating period; sacrifice of parental females occurred after FIB or F2B litters were weaned. Histopathologic evaluation was conducted on the following tissues from all male and female adults from the control and high dose groups after fixation in buffered neutral 10 % formalin, paraffin embedment, sectioning, and staining with hematoxylin and eosin: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, and other tissues with gross lesions. Any of the above organs or tissues showing gross alterations were also evaluated microscopically in the low and mid dose groups.
A complete necropsy was conducted on any parental animal dying on test. The fixed (buffered neutral 10 % formalin) uteri from any female of the F0 or Fl generations failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy status; implantation sites (if any) were recorded. This procedure did not affect any subsequent histopathologic examination of the uteri. The fixed testes were examined histologically for all F0 and Fl adult males which did not sire litters.

Postmortem examinations (offspring):

A gross internal examination was also performed on any pup appearing -abnormal or dying on test, and ten pups randomly selected for each sex from each test group of the F1A, F1B, F2A and F2B generations. Gross findings, if any, from necropsy of the randomly selected weanlings were communicated to the Sponsor's Representative, and at that time the decision was made as to whether remaining pups were to be necropsied or euthanized and discarded.

Statistics:

The results of the quantitative continuous variables (e.g., body weights, food consumption, etc.) were intercompared for the three treatment groups and one control group by use of Leaven’s test for equal variances, analysis of variance and (pooled or separate variance) t-test. Non-parametric data were statistically evaluated using the Kruskall-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate. Frequency data were compared using the Fisher’s exact test.

Reproductive indices:

- Mating index (females)
- Mating index (males)
- Fertility index (female)
- Fertility index (male)
- Number of males siring litters X 100
- Number of males impregnating females

Offspring viability indices:

- Gestational index
- Live birth index
- Number of live pups at birth
- Total number of pups born
- 4-Day survival index
- 7-Day survival index
- 14-Day survival index
- 21-Day survival index
- Lactation index
- 28-Day survival index

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details below

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see details below

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

F0 PARENTAL ANIMALS
- Clinical Signs: There were no significant clinical signs of toxicity observed in F0 males or females.
- Body Weights: Throughout the entire treatment period, body weights of F0 males were reduced at 1500 ppm. Reductions were confirmed statistically on Weeks 1 through 10 of the pre-breed period, and Weeks 11 and 15 after commencement of the mating period. At Week 2 of treatment, body weights at 750 ppm were statistically reduced; otherwise, male body weights at 750 ppm were equivalent to control values. Weight gain reductions were observed throughout the treatment period at 1500 ppm. At 750 ppm, weight gain reductions observed for the first two treatment weeks are consistent with transiently reduced body weights and most likely resulted from reduced palatability of the mid dose diets. At 300 ppm, there was no effect of test chemical administration on body weight or body weight gain.
Body weights of F0 females were consistently reduced at 1500 ppm throughout the pre-breed period and through the interim period between the first and second breeds. These reductions were statistically confirmed on most occasions. At 750 and 300 ppm, F0 female body weights were equivalent to control values. Substantial reductions in body weight gain at 1500 ppm (31 and absolute body weights for all subsequent weeks regardless of compensatory or equivalent weight gains. Body weight gains in the low and mid dose groups were unaffected by test chemical treatment.
- Food Consumption: Food consumption in F0 males was reduced at 1500 ppm throughout the pre-breed period. Reduced food consumption observed at 750 ppm for the first two treatment weeks was considered to be a function of reduced palatability rather than of the treatment per se. Food consumption was unaffected by treatment at 300 ppm.
As expected, because of relatively constant feed consumption and increasing body weight with age, the calculated mean doses of test chemical dropped steadily from Week 1 to 10 in all treated groups: from 32.1 to 16.4 mg/kg bw/day at 300 ppm, from 78.7 to 39.2 mg/kg bw/day at 750 ppm and from 148.8 to 81.0 mg/kg bw/day at 1500 ppm.
Food consumption in F0 females was also significantly decreased at 1500 ppm throughout the pre-breed period. At 300 and 750 ppm, F0 female food consumption was unaffected by test chemical ingestion.
The calculated dosage of test chemical ingested by F0 females exhibited a pattern similar to that of the F0 males wherein dosages ingested dropped from 33.5 to 19.8 mg/kg bw/day at 300 ppm, from 82.0 to 49.9 mg/kg bw/day at 750 ppm and from 148.7 to 102.5 mg/kg bw/day at 1500 ppm.

F0 MATING AND REPRODUCTIVE EFFECTS AT F1A BREED
- Reproductive Parameters: Following the 10-week pre-breed period, the F0 animals were randomly paired within dose groups for 21 days to produce the F1A generation. Gestational length as well as mating, fertility and gestational indices were unaffected by treatment.
- Gestational Body Weights and Food Consumption: Maternal F0 gestational body weights were reduced at 1500 ppm during the gestational period and were confirmed statistically for Gestational Days (gd) 0, 6 and 15. These reductions in gestational body weight are consistent with the 6 % reduction in absolute body weight observed at commencement of the breeding period. Gestational body weight gains at 1500 were equivalent to control values. Body weights and weight gains at 300 and 750 ppm were essentially equivalent to control values throughout gestation. Gestational food consumption was unaffected by test chemical treatment.
- Lactational Body Weights and Food Consumption: Maternal lactational body weights were reduced at 1500 ppm through Lactational Day 14. Increased lactational body weight gain, observed on Postnatal Days 0 to 21 at 1500 ppm, appeared to be compensatory (following consistently reduced body weight during the gestation and pre-breed periods) and was not considered to be an effect of treatment. No significant changes in lactational body weight or lactational weight gain were observed in the 300 or 750 ppm dose groups. Food consumption during F1A lactation was equivalent across dose groups.

F0 MATING AND REPRODUCTIVE EFFECTS AT F1B BREED
- Reproductive Parameters: Approximately ten (10) days after the weaning of the F1A litters, the F0 parents were mated a second time to produce F1B litters. For the F1B breed, F0 females were randomly paired within dose groups to males other than those assigned to them for the F1A breed. Reproductive parameters in the F1B breed were unaffected by treatment. When reproductive parameters for F0 parents were assessed for combined F1A and F1B breeds, there were no differences across groups.
- Gestational Body Weights and Food Consumption: Although not confirmed statistically, gestational body weights and weight gains at 1500 ppm appear lower than control values, indicating continued weight reduction at the high dose level. There were no changes in maternal body weight or body weight gain at 750 or 300 ppm during gestation. There were no treatment-related changes in food consumption during F1B gestation.
- Lactational Body Weights and Food Consumption: Lactational body weight at 1500 ppm was reduced during the first week of lactation. However, substantial compensatory weight gains were observed throughout lactation at 1500 ppm . There were no apparent effects of treatment on lactational body weights at 750 and 300 ppm. Lactational food consumption was equivalent across dose groups .

NECROPSY AND HISTOPATHOLOGY OF F0 PARENTAL ANIMALS
After the weaning of the F1B litters, all of the F0 parental females were necropsied, and histopathology was performed on selected organs from high dose and control animals. F0 parental males were necropsied following the mating period. There were no treatment-related lesions observed at necropsy or in the histopathologic examination of selected tissues.
On Day 28, one week after weaning, 28 F1B pups/sex were randomly selected from each treatment group to become the F1 parents of the next generation.

F1 PARENTAL ANIMALS
- Clinical Signs: Clinical signs observed during the 10-week pre-breed treatment of F1 animals are indicated. None were considered to be treatment related.
- Body Weights: Males at 1500 ppm exhibited consistently reduced body weights throughout the entire treatment period. The reductions were confirmed statistically from pre-breed through the mating period. No body weight depression was noted at 750 ppm, confirming the assumption that the transient weight depression noted in the F0 males during the first two weeks of diet administration was due to diet avoidance. There was no effect of test diet administration at 300 ppm. Significant weight gain depression was noted at 1500 ppm. While increased or equivalent weight gains were also observed at 1500 ppm substantial weight gain depression early in the pre-breed period resulted in reduced body weight values throughout treatment. No treatment-related effects on body weight gain were observed at 750 or 300 ppm.
The F1 adult females exhibited reduced body weights at 1500 ppm for all time points monitored during the pre-breed treatment period. Weight gain depression was noted at 1500 ppm for several time points throughout the pre-breed period and confirmed statistically at pre-breed Weeks 1 and 9. There were no significant effects on body weight or body weight gain at 750 and 300 ppm. (It is unlikely that the statistical reduction in body weight gain for Week 0-1 at 750 ppm is treatment related since mean body weights for the 750 ppm group were slightly above control values at Week 0.)
- Food Consumption: Food consumption for the F1 males was reduced at 1500 ppm throughout the treatment period and was confirmed statistically for the first seven pre-breed treatment weeks. No treatment-related effects on food consumption were observed at 750 or 300 ppm.
The calculated dosage of test chemical ingested in mg/kg dropped over time within groups, from 26.6 to 15.7 at 300 ppm, from 65.8 to 37.9 at 750 ppm and from 140.7 to 78.7 mg/kg at 1500 ppm.
The F1 females exhibited reductions in food consumption at 1500 ppm for the entire 10 pre-breed period. No effects on food consumption were noted in F1 females at 750 and 350 ppm.
Calculated dosages of test chemical ingested over time for the females were: 30.1 to 19.7 mg/kg at 300 ppm, 73.5 to 48.9 mg/kg at 750 ppm and 153.9 to 99.4 mg/kg at 1500 ppm.

F1 MATING AND REPRODUCTIVE EFFECTS AT F2A BREED
- Reproductive Parameters: After the completion of the 10-week pre-breed period, the F1 animals were randomly paired within dose groups to produce the F2A generation. Reproductive parameters for the F2A breed are presented in Table 41. No reproductive parameters including mating and fertility indices and gestational length were affected by treatment.
- Gestational Body Weights and Food Consumption: Maternal F1 body weights were reduced during gestation in the 1500 ppm dose group. While gestational weight gains were equivalent to control values, overall body weight reductions at 1500 ppm are consistent with significantly reduced body weights noted prior to the breeding period. Gestational body weights and body weight gains at 750 and 300 ppm were equivalent to control values. Food consumption
during F2A gestation was unaffected by test chemical administration.
- Lactational Body Weights and Food Consumption: Body weights were reduced at 1500 ppm for the first week of lactation. Compensatory weight gains were observed at 1500 ppm following delivery of litters at a reduced body weight. Body weights and weight gains at 750 and 300 ppm were unaffected by treatment during lactation. There were no significant effects of test chemical administration on food consumption during lactation.

F1 MATING AND REPRODUCTIVE EFFECTS AT F2B BREED
- Reproductive Parameters: Approximately ten (10) days after the weaning of the F2A litters, the F1 adults were paired a second time to produce the F2B generation. For the F2B breed, F1 females were randomly paired within dose groups to males other than those assigned to them for the F2A breed. Reproductive parameters for the F2B breed were unaffected by treatment. Reproductive parameters assessed for both F2A and F2B breeds demonstrated no reproductive effects due to test chemical ingestion.
- Gestational Body Weights and Food Consumption: For the F2B breed, maternal body weights at 1500 ppm were reduced throughout gestation. While body weight changes at 1500 ppm were equivalent to control values, reduced body weights are consistent with substantial reductions observed prior to the mating period. There were no treatment-related effects on gestational body weights or weight gains at 750 and 300 ppm for the entire gestational period. Food
consumption during gestation was unaffected by test chemical administration.
- Lactational Body Weights and Food Consumption: Lactational body weights were reduced at 1500 ppm through Lactational Day 14. As in the F2A breed, compensatory weight gains were observed at 1500 ppm following delivery of litters at a reduced weight. No effects on body weight were noted at 750 and 300 ppm. Food consumption during lactation was unaffected by treatment.

NECROPSY AND HISTOPATHOLOGY OF F1 PARENTAL ANIMALS
After weaning of the F2B litters, all of the F1 parental females were subjected to necropsy, and histopathologic examination of selected tissues was conducted on high dose and control animals. F1 adult males were necropsied following the mating period. No treatment-related lesions were observed at necropsy or in the histopathologic examinations.

Dose descriptor:

NOAEL

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details below

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

F1A PUPS
- Litter Size and Sex Ratio: F1A litter sizes and sex ratios (% males) were unaffected by treatment.
- Body Weights: Pup body weights and body weight gains (by litter and by sex by litter) were unaffected by treatment through Lactational Day 7. From Day 14 through weaning on Lactational Day 21, and postweaning, on Day 28, body weights of male and female pups at 1500 ppm were reduced. Corresponding body weight gains for Lactational Days 7-21 and 21-28 (postweaning) were reduced at 1500 ppm as well. Body weights and weight gains of pups at 750 and 300 ppm were unaffected by treatment.
- Viability and Survival: The numbers of pups which died during the lactation period (Postnatal Days 0 to 4, precull, and Postnatal Days 7-14, 4-14, 14-21 and 4-21) were equivalent across all groups. Consistent with these observations, F1A live birth and survival indices were equivalent across all dose groups for the lactation (Postnatal Days 0-21) and postweaning periods (Postnatal Days 0-21 and 21-28, respectively).
- F1A Pup Necropsy: No treatment-related lesions were observed in the F1A pups which died during the lactation period. Necropsy of ten (10) randomly selected F1A weanlings/sex/dose group indicated no treatment-related findings.

F1B PUPS
- Litter Size and Sex Ratio: F1B litter sizes and sex ratios (% males) were equivalent across all groups.
- Body Weights: Mean F1B pup body weights and body weight gains per litter (total, male and female) were unaffected by treatment through Lactational Day 7. From Day 14 through Days 21 and 28 (postweaning), pup body weights per litter were reduced at 1500 ppm. Corresponding pup body weight gains per litter (entire, male and female) were reduced for Lactational Days 7-21 and 21-28 at 1500 ppm. F1B pup body weights and weight gains at 750 and 300 ppm were unaffected by treatment.
- Viability and Survival: There were no treatment-related effects on the numbers of pups which died in the postnatal period; F1B pup survival indices were, therefore, unaffected by treatment.
- F1B Pup Necropsy: No treatment-related lesions were observed in F1B pups which died during the lactational period. Necropsy of ten (10) randomly selected F1B pups/sex/dose group indicated no treatment-related findings.

F2A Pups
- Litter Size and Sex Ratio: F2A litter sizes and sex ratios (% males) were unaffected by treatment.
- Body Weights: F2A pup body weights were equivalent across dose groups from birth through Lactational Day 14. At weaning, and postweaning on Lactational Day 28, pup body weights per litter (males, females and all pups) were reduced at 1500 ppm. Coincident pup body weight gains per litter from Lactational Days 14-28 were also reduced at 1500 ppm. There were no treatment-related effects on F2A pup weights or weight gains at 750 and 300 ppm.
- Viability and Survival: No increases in pup deaths were observed during lactation in any dose groups relative to the control group (Table 48). F2A pup survival indices were consequently equivalent across all dose groups.
- F2A Pup Necropsy: No treatment-related lesions were observed in F2A pups which died during lactation, or in the ten weanlings/sex/dose subjected to gross necropsy.

F2B PUPS
- Litter Size and Sex Ratio: There was no effect of treatment on F2B litter size from birth through Lactational Days 21 (The statistically increased sex ratio (% males) observed prior to culling at 750 and 1500 ppm was considered to be due to random variation).
- Body Weights: F2B pup body weights per litter (males, females, and total) were equivalent through Lactational Day 4. From Lactational Day 7 through weaning on Day 21, and postweaning, on Day 28, body weights of F2B pups at 1500 ppm were reduced. Note that slight reductions in body weight which achieved statistical significance only in female pups on Day 14 were also apparent on Day 7. Concurrent F2B pup weight gain reductions were observed at 1500 ppm from Day 4-28. There were no observable effects of treatment on body weight at 750 and 300 ppm.
- Viability and Survival: There was no effect of test substance administration on the number of stillborn F2B pups or on the number of pups which died from birth through Postnatal Day 28. Consequently, F2B pup survival-indices throughout lactation were equivalent across all dose groups.
- F2B Pup Necropsy: No treatment-related lesions were observed in the examination of F2B pups which died during the lactation period, or in the ten F2B weanlings/sex/dose subjected to gross necropsy after weaning.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Reproductive effects observed:

not specified

Conclusions:

These results indicate that continuous exposure to test item in the diet for two generations in Sprague-Dawley (CD®) rats resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm, limited to body weight reduction, weight gain depression and decreased food consumption. Postnatal toxicity was observed at 1500 ppm, consisted of reduced pup body weights. The adult effect levels are equivalent to 112.6 mg of test chemical per kg body weight at 1500 ppm. The "no observable effect level" (NOEL) for adults in this study was 750 ppm. The NOEL for offspring in this study was 750 ppm (males: 38 - 79 mg/kg bw/day and females: 49 - 82 mg/kg bw/day), indicating no increased risk to offspring in the absence of maternal effects.

Executive summary:

A study was carried out according to EPA OPP 83-4 (Reproduction and Fertility Effects) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate. Male and female CD® (Sprague-Dawley) weanling rats (the F0 generation) were exposed to test item by dietary inclusion at target dosage levels of 0, 300, 750 or 1500 ppm for two generations. A total of 28 males and 28 females were evaluated at each dosage level. Following a 10-week pre-breed exposure period, the F0 rats were randomly paired within dose groups for a three (3)-week mating period to produce the F1A generation. Exposures continued through mating, gestation, parturition and lactation. At least ten (10) days after the weaning of the F1A litters, the F0 parents were paired again (with different male-female pairing within the dose groups than employed for the F1A breed) for three (3) weeks to produce the F1B generation.

Exposures continued through mating, gestation, parturition and lactation of the second litters. After the F1B pups were weaned, the F0 animals were necropsied and high dose and control animals examined for histopathologic lesions. At weaning, twenty-eight (28) F1B weanlings/sex/group were randomly selected as parents of the subsequent generation. In addition, ten (10) F1B weanlings/sex/dose were randomly selected for necropsy and examination of gross lesions. Selected F1 parents were exposed to the same dietary concentrations of test item as their parents for at least ten (10) weeks. After their pre-breed exposure the F1 animals were paired as described above to produce the F2A and F2B offspring. Mating, gestation, lactation and necropsy of the F1 parents and selected F2A and F2B weanlings were performed as described above except that no F2 animals were selected as parents. Remaining non-selected F1A, F1B, F2A and F2B pups at weaning were euthanized and discarded after review of the necropsy of their selected littermates.

During the 10-week pre-breed period, no significant clinical signs of toxicity were observed in F0 males or females. F0 males and females at 1500 ppm exhibited consistently reduced body weights and weight gain beginning one week after treatment. Substantial reductions in food consumption were also observed throughout the pre-breed period in F0 males and females at 1500 ppm.

During the first two weeks of exposure, body weights and food consumption also were slightly lower in F0 male rats at 750 ppm. These transient depressions were attributed to initial diet avoidance rather than to a treatment-related toxic effect. There were no treatment-related effects on body weight or food consumption in F0 females at 750 ppm or in F0 males and females at 300 ppm.

At the F0 breed to produce F1A litters reproductive parameters including mating and fertility indices and gestational length were unaffected by treatment. There were significant reductions in gestational body weights but not body weight gain at 1500 ppm; lactational body weights but not body weight gains were reduced for the first two weeks of lactation at 1500 ppm. No effects of treatment on gestational body weight or weight gains were noted at 750 and 300 ppm. Food consumption was unaffected throughout gestation and lactation.

During the second breeding of the F0 animals to produce F1B litters, there was no effect of treatment on reproductive parameters. Gestational body weights and weight gains at 1500 ppm appeared to be reduced. Lactational body weights but not weight gains were reduced at 1500 ppm. No effects of treatment on body weights were observed during gestation at 750 and 300 ppm. Food consumption was unaffected throughout F1B gestation and lactation.

At necropsy of F0 adults, no treatment-related lesions were observed. No treatment-related lesions were observed in the histopathologic examination of selected organs from high dose and control F0 adults.

The F1A litters from the first breeding of F0 adults exhibited reduced body weights from Postnatal Days 14 through 28 at 1500 ppm. Corresponding F1A pup weight gains at 1500 ppm were reduced for Lactational Days 7-21 and 21-28. Body weights and weight gains of F1A litters at 750 and 300 ppm were unaffected by treatment. There was no effect of treatment on postnatal deaths (Postnatal Days 0-28) of F1A pups.

F1B pups from the second breeding of F0 adults exhibited reduced body weights from Postnatal Days 14 through 28 at 1500 ppm; pup weight gains for Days 7-21 and 21-28 were reduced at 1500 ppm.

There was no effect of treatment on body weights or weight gains in F1B pups at 750 and 300 ppm. Postnatal survival of F1B pups was unaffected by treatment. There were no treatment-related lesions observed in the necropsy of F1A and F1B pups which died during lactation or of randomly selected F1A and F1B weanlings (ten/sex/dose).

During the 10-week pre-breed exposure of the second (F1) generation animals, no significant clinical signs of toxicity were observed. F1 males and females at 1500 ppm exhibited consistently reduced body weights and substantially reduced body weight gain during the pre-breed period. Food consumption was also reduced in F1 males and females at 1500 ppm. There were no treatment-related effects on body weight, weight gain or food consumption at 750 and 300 ppm.

At the F1 breed to produce F2A litters, reproductive parameters were unaffected by treatment. Maternal gestational body weights but not weight gains were reduced at 1500 ppm. Lactational body weights but not weight gains at 1500 ppm were reduced for the first week of lactation. There were no body weight effects of treatment at 750 and 300 ppm. Food consumption during gestation and lactation was unaffected by treatment.

During the second breeding of the F1 animals to produce F2B litters reproductive parameters remained unaffected by treatment. Maternal gestational body weights but not weight gains at 1500 ppm were reduced. Lactational body weights but not weight gains at 1500 ppm were reduced for the first two weeks of the lactational period. There were no treatment-related effects on body weight at 750 and 300 ppm. Food consumption during F2B gestation and lactation was unaffected by treatment.

At necropsy of F1 adults, no treatment-related lesions were observed. There were also no treatment-related lesions observed in the histopathologic examination of selected organs from high dose and control F1 adults. There were no treatment-related deaths of adult animals on study.

F2A pup body weights per litter were reduced at 1500 ppm on Postnatal Days 21 and 28. Concurrent weight gains for Days 14-28 were reduced at 1500 ppm. F2A pup weights were unaffected by treatment at 750 and 300 ppm. Perinatal deaths and survival of F2A pups were unaffected by treatment.

Body weights per litter of F2B pups were reduced at 1500 ppm for Postnatal Days 7 through 28. Pup weight gains for Postnatal Days 4-21 and 21-28 were also reduced at 1500 ppm. No effect of treatment on F2B pup body weights was observed at 750 and 300 ppm. F2B pup survival was unaffected by treatment.

There were no treatment-related lesions observed at necropsy of F2A and F2B pups which died during lactation or of randomly selected F2A and F2B weanlings (ten/sex/dose).

In conclusion, exposure to test item in the diet for two generations at doses of 1500, 750 and 300 ppm resulted in well-defined parental and postnatal toxicity at the 1500 ppm dose level without any adverse effects on reproductive performance. The "no observed effect level" (NOEL) for adults in this study was 750 ppm (males: 38 - 79 mg/kg bw/day and females: 49 - 82 mg/kg bw/day); the NOEL for offspring was 750 ppm, indicating no increased risk to the offspring in the absence of indications of adult toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

38 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Good quality database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
A study was carried out according to EPA OPP 83-4 (Reproduction and Fertility Effects) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate. The results indicate that continuous exposure to test item in the diet for two generations in Sprague-Dawley (CD®) rats resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm, limited to body weight reduction, weight gain depression and decreased food consumption. Postnatal toxicity was observed at 1500 ppm, consisted of reduced pup body weights. The adult effect levels are equivalent to 112.6 mg of test chemical per kg body weight at 1500 ppm. The "no observed effect level" (NOEL) for adults in this study was 750 ppm. The NOEL for offspring in this study was 750 ppm (males: 38 - 79 mg/kg bw/day and females: 49 - 82 mg/kg bw/day), indicating no increased risk to offspring in the absence of maternal effects.

Justification for selection of Effect on fertility via oral route:
- Reliability: GLP study according to EU- and/or OECD-guidelines
- Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Effects on developmental toxicity

Description of key information

A key and a supporting study were carried out according to OECD Guideline 414 and EPA OPP 83-3 (Prenatal Developmental Toxicity Study) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate.
In the key study test item administered by gavage during organogenesis to CD® (Sprague-Dawley) rats produced characteristic clinical signs of maternal toxicity (rapid respiration and gasping) at 10.0 and 20.0 mg/kg bw/day and reductions in body weight and food consumption at 20.0 mg/kg bw/day. No evidence of developmental toxicity including teratogenicity was observed at any dose employed. The A/D ratio (the ratio of the adult lowest observable effect level to the developmental lowest observable effect level) is less than 1 (10 mg/kg bw/day:at least 20 mg/kg bw/day) indicating no preferential susceptibility of the rat conceptus to the test substance under the conditions of this study.
The "no observable effect level" for maternal toxicity was 1.0 mg/kg bw/day; the NOEL for developmental toxicity was at least 20.0 mg/kg bw/day.
In the supporting study test item administered by gavage during organogenesis in New Zealand white rabbits produced indications of maternal toxicity including mortality at 10.0 mg/kg bw/day, and nonlethal indications of maternal toxicity at 3.0 and 10.0 mg/kg bw/day, including reduced weight gain and clinical signs during the treatment period. Developmental toxicity, expressed as increased incidence of dead fetuses per litter and reduced fetal body weights per litter, was observed at 10.0 mg/kg bw/day. There was no evidence of teratogenicity at any dose employed. The NOEL for maternal toxicity was 1.0 mg/kg bw/day; the NOEL for developmental toxicity was 3.0 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Reliability: GLP study according to international guidelines - Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: males 250 to 300 g; females 175 to 200
- Housing: individually in stainless steel wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: ca. two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66 to 77
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12 hrs

IN-LIFE DATES:
- From: 05 February 1990
- To: 01 March 1990

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): twice during the study

VEHICLE
- Concentration in vehicle: 0.2, 2.0 and 4.0 mg/mL
- Amount of vehicle: 5.0 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Standard solutions used for analyses were prepared as follows. A standard stock solution (1.0 mg/mL) was prepared by weighing 0.0619 g of test substance into a 50 mL volumetric flask and diluting to volume with acetonitrile. The 1.0 mg/mL standard was then appropriately diluted with acetonitrile to generate standard solutions of 10, 50 and 100 ng/μL. An aliquot of each dosing formulation was diluted with acetonitrile as described above. One (1.0) μL of each diluted dosing solution was analyzed for test substance content. All dosing samples were analyzed with a Hewlett-Packard 5880A Gas Chromatograph equipped with a Nitrogen Phosphorous detector. The formulations and analyses were based on actual test item content.
The dosing solutions were homogeneous, stable for at least 12 days when stored at room temperature, and within 97.0 to 103.8 % of nominal. Formulations were based on actual test item content of the test material. No test substance was detected in the control solutions at minimum detection limit of approximately 0.01 mg/mL.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: 05 to 08 Februar 1990
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug

Duration of treatment / exposure:

Timed-pregnant CD® (Sprague-Dawley) rat dams Were dosed daily with test item in vehicle or vehicle alone (Millipore® water) on gd 6 through 15. All treatments were administered by gavage using a 16 gauge stainless steel dosing tube, 3.0 inches long (Perfektum®, Popper and Sons, Inc., New Hyde Park, NY), attached to a 3.0 cc disposable syringe. The dose volume employed was 5.0 mL/kg body weight. Administered volumes were adjusted on the basis of the most recent body weight of each animal.
All females on study were weighed on gd 0, 6 (prior to onset of dosing), 9, 12, 15 (during the dosing period), 18 and 21. Food consumption was measured at three-day intervals throughout gestation (gd 0-21). All females were thoroughly examined daily for clinical signs of toxicity (twice daily during the dosing period). In addition, the animals were examined twice daily for mortality and morbidity.

Frequency of treatment:

Once daily during exposure period

Duration of test:

Days 6-15 of gestation

Remarks:

Doses / Concentrations:
0, 1, 10 and 20 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 0.0, 1.0, 10.0 and 20.0 mg/kg bw/day were selected based on information from a dose range-finding study on pregnant rats

Maternal examinations:

The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic organs and cavities were examined grossly. The lumen and lining of the esophagus, stomach and trachea were examined for any indications of irritation from the dosing solutions and for dosing errors. No females exhibited evidence of dosing error.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during dosing, daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 15, 18 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Three day intervals

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: All surviving study females were sacrificed on gd 21 by carbon dioxide asphyxiation. The sacrifice period was February 27 through March 1, 1990.
- Organs examined: The maternal body cavities were opened by a mid-sagittal thoracolaparotomy. The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic organs and cavities were examined grossly. The lumen and lining of the esophagus, stomach and trachea were examined for any indications of irritation from the dosing solutions and for dosing errors. No females exhibited evidence of dosing error.

Ovaries and uterine content:

Ovarian corpora lutea of pregnancy were counted. The uterus was externally examined for signs of hemorrhage, removed from the abdominal cavity, weighed, and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were noted and recorded. Uteri from females that appeared nongravid were placed in a 10 % ammonium sulfide solution for detection of early resorptions (Salewski, 1964). Maternal liver weights also were determined.

Fetal examinations:

All live fetuses were weighed, sexed, examined for external malformations including cleft palate, and variations. Approximately one half of the fetuses in each litter (even-numbered fetuses from litters with an even number of live fetuses, odd-numbered fetuses from litters with an odd number of liver fetuses) were examined for visceral (thoracic and abdominal) abnormalities by modification of methods described by Staples (1974). These fetuses were then decapitated and their heads fixed in Bouin's solution for examination of craniofacial structures by sectioning methods modified from Wilson (1965, 1973). All sectioned heads were saved after examination. The remaining half of fetuses in each litter (intact) were eviscerated and processed for skeletal staining with alizarin red S (Crary, 1962) and examined for skeletal malformations and variations. Following skeletal examination, skeletal preparations from all fetuses were bagged individually and all fetuses within each litter were bagged together. The skeletons of the fetuses subjected to visceral examinations and decapitated Were also stained and stored, but were not examined.

Statistics:

Levene’s test for equal variances, analysis of variance, and t-tests with Bonferroni probabilities for pairwise comparisons. Nonparametric data were analyzed with Kruskal-Wallis test followed by Mann-Whitney U test when appropriate. Incidence data were compared using Fisher’s Exact Test.

Indices:

No data

Historical control data:

Historical data on 2,452 control CD® litters have been summarized across teratology studies from nine pharmaceutical companies or research organizations by the Middle Atlantic Reproduction and Teratology Association (Woo and Hoar, 1979).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- Clinical signs: Audible respiration and gasping at 20 mg/kg bw/day. Audible respiration at 10 mg/kg bw/day
- Mortality: No mortalities
- Body weight gain: Reduced body weight gain at 20 mg/kg bw/day
- Food consumption: Reduced food consumption at 20 mg/kg bw/day
- Gross findings at necropsy: Ulceration of stomach and gas filled intestines at 20 mg/kg bw/day. Gravid uterine and liver unaffected.
- Other: No abortions or early births

No dams died, aborted, delivered early or were removed from the study. At scheduled laparotomy one (1) dam each at 1.0 and 10.0 mg/kg bw/day and two (2) dams at 20.0 mg/kg bw/day were not pregnant. All pregnant dams had one or more live fetuses at scheduled sacrifice (no litters were fully resorbed).
Twenty-three (23) to 25 litters per group were available for examination at scheduled sacrifice. All subsequent presentation and discussion of summary data are based on pregnant animals.
Treatment-related clinical signs of toxicity observed during the treatment period included audible respiration and gasping at 20.0 mg/kg bw/day and audible respiration at 10.0 mg/kg bw/day. At 20.0 mg/kg bw/day, audible respiration was observed subsequent to the treatment period as well.
While not statistically significant, there appears to be a treatment-related reduction in maternal body weight for Days 12, 15, 18 and 21 at 20 mg/kg bw/day. Body weight gains also appeared to be decreased for Days 9 to 12, 12 to 15 and 6-15 at 20 mg/kg bw/day, and body weight gains appear reduced for the entire gestational period, Day 0-21. There were no apparent changes in body weight or weight gain at 10.0 and 1.0 mg/kg bw/day. Apparent reductions in food consumption at 20 mg/kg bw/day for Days 9 to 12, 12 to 15 and 6 to 15 correlate well with reductions in body weight and severity of clinical signs during the treatment period. Food consumption at 10.0 and 1.0 mg/kg bw/day was equivalent across groups during and subsequent to the treatment period.
Necropsy observations in dams sacrificed on gd 21 were recorded. Treatment-related findings included ulceration of the stomach and gas-filled intestines at 20.0 mg/kg bw/day.
Slight treatment-related reductions on maternal terminal body weight corrected weight change were observed at 20.0 mg/kg bw/day. Gravid uterine weight and liver weight were unaffected by treatment.
Gestational parameters including the number of corpora lutea, total implants, viable and nonviable implants, pre- and post implantation loss and sex ratio (% male fetuses) per litter were equivalent among groups. Fetal body weight per litter was unaffected by test substance treatment.

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Bodyweight: No treatment-related effects
- Gross findings at necropsy: No malformations
- Skeletal findings: No treatment-related variations or malformations
- Visceral findings: No treatment-related variations or malformations

There were no treatment-related increases in individual external, visceral or skeletal fetal malformations, in malformations by category or in total malformations in this study. There were no differences in the incidences of external and visceral variations were observed between control and treated groups. The incidence of two skeletal variations, poorly ossified cervical centrum #7 and majority of proximal hindlimb phalanges unossified, were significantly decreased in the 10.0 and 20.0 mg/kg bw/day dose groups, respectively. These reduced incidences were not considered to be related to test substance administration. The incidences of variations by category (external, skeletal or visceral) and total variations were not significantly altered by test substance administration.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Test item administered by gavage during organogenesis to CD® (Sprague-Dawley) rats produced characteristic clinical signs of maternal toxicity (rapid respiration and gasping) at 10.0 and 20.0 mg/kg bw/day and reductions in body weight and food consumption at 20.0 mg/kg bw/day. No evidence of developmental toxicity including teratogenicity was observed at any dose employed. The A/D ratio (the ratio of the adult lowest observable effect level to the developmental lowest observable effect level) is less than 1 (10 mg/kg bw/day:at least 20 mg/kg bw/day) indicating no preferential susceptibility of the rat conceptus to the test substance under the conditions of this study.
The "no observable effect level" for maternal toxicity was 1.0 mg/kg bw/day; the NOEL for developmental toxicity was at least 20.0 mg/kg bw/day.

Executive summary:

A study was carried out according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) and EPA OPP 83-3 (Prenatal Developmental Toxicity Study) using the structural analog Didecyldimethylammonium chloride (DDAC). In view of the chemical and structural similarities (the relevant chemical part of both, DDAC and DDACarbonate, under the conditions of this test is the common quaternary ammonium cation Didecyldimethylammonium+), it is considered that the data are adequate for DDACarbonate. Timed-pregnant CD® (Sprague-Dawley) rats were exposed to test item in deionized water by gavage on gestational days (gd) 6 through 15. Twenty-five (25) copulation plug-positive females per group were dosed with doses of 1.0, 10.0 and 20.0 mg/kg bw/day. The dose volume employed was 5.0 mL/kg of body weight based on the most recent body weight of each female. The vehicle control group (0.0 mg/kg bw/day) of twenty-five females received deionized water at a volume of 5.0 mL/kg bw/day. Clinical observations were taken daily (twice daily during dosing) and maternal body weights were recorded for all study females on gd 0, 6, 9, 12, 15, 18, and 21. Maternal food consumption was measured at three-day intervals throughout gestation, gd 0-21. At scheduled sacrifice on gd 21, the dams were evaluated for body weight, liver and gravid uterine weight, number of corpora lutea and number and status of implantation sites (i.e. early and late resorptions, dead fetuses, live fetuses). All live fetuses were dissected from the uterus, counted, sexed, weighed, and examined for external maformations (including cleft palate) and variations. Approximately one-half of the fetuses in each litter were examined for visceral (including craniofacial) malformations and variations. The remaining one-half of the fetuses were evaluated for skeletal malformations and variations. No females died, aborted, delivered early or were removed from study. Pregnancy rate was approximately equivalent across groups ranging from 92 % to 100 %. All surviving pregnant females had one or more live fetuses at scheduled sacrifice, and 23 to 25 litters were examined at each dose. Maternal toxicity was indicated at 10.0 and 20.0 mg/kg bw/day by characteristic clinical signs of audible respiration. At 20 mg/kg bw/day, gasping was observed during the dosing period and audible respiration persisted subsequent to treatment. Reductions in body weight and food consumption were observed at 20.0 mg/kg bw/day during the treatment period. Maternal terminal body weight and corrected weight change were reduced at 20.0 mg/kg bw/day. There were no effects on maternal gravid uterine weight, liver weight or relative liver weight. Treatment-related necropsy findings in dams at scheduled sacrifice included ulceration of the stomach and gas-filled intestines at 20.0 mg/kg bw/day. Gestational parameters, including ovarian corpora 1utea of pregnancy, implantations per litter (viable, nonviable and total), sex ratio and fetal body weight per litter were not affected by treatment. There were no significant treatment-related external, visceral or skeletal malformations observed in this study. There were no increased incidences of external, visceral or skeletal fetal variations. Administration of didecy1dimethy1ammoniumch1oride by gavage to CD® (Sprague-Dawley) rats during organogenesis resulted in characteristic signs of maternal toxicity at 10.0 and 20.0 mg/kg bw/day including audible respiration and gasping during the treatment period at 10.0 and 20.0 mg/kg bw/day and reduced body weight and food consumption at 20.0 mg/kg bw/day. No treatment-related developmental toxicity, including teratogenicity, was observed at any dose 1eve1. The "no observed effect level" (NOEL) for maternal toxicity was 1.0 mg/kg bw/day. The NOEL for developmental toxicity was at least 20.0 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality database

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
- Reliability: GLP study according to international guidelines
- Read-across justification: As both source (Didecyldimethylammonium chloride, DDAC) and target chemicals (Didecyldimethylammonium carbonate, DDA carbonate) have identical organic cations with hydrophobic side chains - the only difference is the inorganic anion carbonate or chloride with negligible contribution to the hazard properties - both substances can be predicted to have similar movement and fate characteristics.

Justification for classification or non-classification

Based on the data available the substance is not classified and labeled according to Regulation (EC) No 1272/2008 (CLP) or Directive 67/548/EEC (DSD).