Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without deficiencies
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
yes
Remarks:
Minor deviations occurred. The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Description: Brown highly viscous liquid
Batch: 966018
Purity: UVCB 100%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: Upon receipt of the animals.
Identification F0: Earmark and tattoo.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Exposure period: Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 44 (Group 1), 65, (Group 3) and 77 (Group 4) were not dosed during littering.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted according to a validated method. These analyses were conducted after the in-life phase as no suitable analytical method was available at an earlier stage. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Female nos. 74 and 75 were re-mated with a proven male of the same dose group since male nos. 34 and 35 were sacrificed in extremis on Day 5 and 2 of the mating period respectively.
Duration of treatment / exposure:
After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 95, 298 and 893 mg/kg/day. Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
No. of animals per sex per dose:
10 per sex per dose level including control

Examinations

Maternal examinations:
Mortality / Viability:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

Clinical signs:
Daily detailed clinical observations were made in all animals immediately (0-15 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Ovaries and uterine content:
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth.
Fetal examinations:
Each litter was examined to determine the following, if practically possible -

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
Indices:
see table attached.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Most females at 893 mg/kg showed hunched posture and piloerection during the post-coitum and/or lactation phase. The female sacrificed at 893 mg/kg additionally showed laboured respiration, red staining of the snout and diarrhoea. At lower incidence, lethargy and lean appearance were noted among surviving females. At 298 mg/kg, the female sacrificed in extremis during the post-coitum phase (no. 66) showed hunched posture, piloerection, red staining of the snout and diarrhoea predominantly during the week preceding death. A few surviving animals incidentally showed hunched posture.
At 95 mg/kg, no toxicologically relevant clinical signs were noted.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
ca. 893 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Mean body weight of pups at 893 mg/kg on Day 4 of lactation was lower than those of control pups (achieving statistical significance for male pups only).
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs and macroscopy) were observed.
The apparent lower gestation index at 893 mg/kg was attributed to the lower number of pregnant females (8) in combination with the higher relative effect on the gestation index of the female (no. 74) that was sacrificed before delivery. Histopathology of this female confirmed presence of a placenta. One female at 893 mg/kg (no. 75) had post-implantation loss (found pregnant based on presence of implantation sites). At 298 mg/kg, of the 10 pregnant females one female (no. 66) was not pregnant and was sacrificed in extremis on Day 23 of the post-coitum phase, and one female (no. 62) had post-implantation loss (found pregnant based on presence of implantation sites). At 95 mg/kg, three females did not become pregnant, and in the control group one female did not become pregnant.
Duration of gestation was similar between the groups.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs and external macroscopy did not reveal treatment-related findings.

Mortality
One pup of the control group, two pups at 95 mg/kg, and two pups at 893 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing/dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs
Incidental clinical symptoms of pups consisted of insufficient milk in the stomach, absence of milk in the stomach and a blue snout or body. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Macroscopy
The nature and incidence of absence of milk in a single surviving pup remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 893 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Developmental toxicity in the form of lower pup body weight in female pups was observed at the high dose of 893 mg/kg on day 4 after birth. There were no significant differences on day 1 in female pups, in male pups on day 1 or day 4 or in combined male and female pup weights on either day 1 or day 4 after birth. No developmental toxicity was observed for the lower treatment groups of 95 and 298 mg/kg body weight/day. Based on the lower female pup weight difference at day 4, the NOAEL was very conservatively set at 298 mg/kg body weight/day. However, clear parental effects were seen at 893 mg/kg bw/d. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.

 
Executive summary:

Di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid(DNNSA) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 95, 298 and 893 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (for 41-52 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Developmental results

Developmental toxicity in the form of lower pup body weight in female pups was observed at the high dose of 893 mg/kg on day 4 after birth. There were no significant differences on day 1 in female pups, in male pups on day 1 or day 4 or in combined male and female pup weights on either day 1 or day 4 after birth. No developmental toxicity was observed for the lower treatment groups of 95 and 298 mg/kg body weight/day. Based on the lower female pup weight difference at day 4, the NOAEL was very conservatively set at 298 mg/kg body weight/day. However, clear parental effects were seen at 893 mg/kg bw/d. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. In addition, most females at 893 mg/kg showed hunched posture and piloerection during the post-coitum and/or lactation phase.Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.

 

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e.gestation index and duration, parturition, maternal care and earlypostnatalpup development consisting of mortality, clinical signs and macroscopy).

 

In conclusion, treatment withdi C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid(DNNSA) by oral gavage in male and female Wistar Han rats at dose levels of 95, 298, 893 mg/kg body weight/day revealed parental toxicity at 298 and 893 mg/kg body weight/day. Developmental toxicity (lower female pup body weights at one time point - day 4) observed at 893 mg/kg/day was considered equivocal and related to maternal toxicity. No reproduction toxicity was observed for treatment up to 893 mg/kg body weight/day.

 

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL:            95 mg/kg/day

Reproduction NOAEL:     893 mg/kg/day

Developmental NOAEL: 893 mg/kg/day