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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: OECD 422 Reproductive Screening Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2012 to 07 December 2012 (end of in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study without significant deficiencies
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
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.

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.
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.
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.
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).

No test substance was detected in the Group 1 formulation.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-54 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. 66 (Group 4) and 75 (Group 4) were not dosed during littering.
Frequency of treatment:
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.
Details on study schedule:
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.
No. of animals per sex per dose:
10 (see table)
Control animals:
yes, concurrent vehicle
Positive control:
No
Parental animals: Observations and examinations:

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.
Oestrous cyclicity (parental animals):

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. 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. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sperm parameters (parental animals):

From the selected 5 males of the control and high dose group (see Allocation), and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
Litter observations:
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.
Postmortem examinations (parental animals):

All males and the selected 5 females/group (see Allocation) were deprived of food overnight (with a maximum of approximately 24.5 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Postmortem examinations (offspring):
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.

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 5) to determine intergroup differences followed by the Wilcoxon test (Ref. 6) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
See tables below
Offspring viability indices:
See tables below
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Parental animals did not show any adverse effects from treatment throughout the study.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Description (incidence and severity):
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.
Histopathological findings:
not examined

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility. Furthermore, the spermatogenic staging profiles were normal for all selected control and high dose males, and for all males suspected to be infertile.
Reproductive effects observed:
not specified

No toxicologically relevant effects on reproductive parameters were noted.

 

The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

 

For female no. 41, the number of pups were slightly higher than the number of implantations and corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 6 of lactation.

Conclusions:
Treatment with Alkylated Naphthalene by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity or reproductive up to 1000 mg/kg.
Based on these results, a parental and reproductive No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Alkylated Naphthalene in rats by oral gavage.

The study was based on the   OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

Rationale for dose levels

Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

 

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 - 54 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).

Results/discussion

 

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

 

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

 

Conclusion

 

Treatment with Alkylated Naphthalene by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity or reproductive toxicity up to the highest dose of 1000 mg/kg.

 

Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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:
No reproduction toxicity was observed for treatment up to 1000 mg/kg body weight/day.

Justification for selection of Effect on fertility via oral route:
GLP study following current guidelines.

Effects on developmental toxicity

Description of key information
No developmental toxicity was observed up to and including the maximum dose of 1000 mg/kg/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October 2012 to 07 December 2012 (end of in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without deficiencies
Reason / purpose:
reference to other study
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
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.
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: Dose volume (mL/kg body weight) were calculated as follows:
Dose level (g/kg) / density (g/mL). Actual dose volumes were calculated according to the latest body weight.
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.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-54 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. 66 (Group 4) and 75 (Group 4) were not dosed during littering.
Frequency of treatment:
Once Daily, 7 days per week.
Duration of test:
Females and pups: 14 October 2012 07 December 2012 (end of in-life phase)
Males: 14 October 2012 12 November 2012 (end of in-life phase)
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 per sex per dose level including control
Control animals:
yes, concurrent no treatment
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.

Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

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.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternal toxicity was observed.
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No embryotoxicity or teratogenic effects observed.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Developmental toxicity was not observed.
For risk assessment purposes the high dose treatment of 1000 mg/kg/day was established as the NOAEL for Alkylated Naphthalene.
 
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Alkylated Naphthalene in rats by oral gavage.

The study was based on the  OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

Rationale for dose levels

Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

 

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 - 54 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).

Results/discussion

 

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

 

Developmental results:

No developmental effects were observed up to the highest dose level tested (1000 mg/kg).

 

Conclusion

 

Treatment with Alkylated Naphthalene by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity or developmental toxicity up to the highest dose of 1000 mg/kg.

 

Based on these results, a parental and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.


Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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
Justification for selection of Effect on developmental toxicity: via oral route:
GLP guideline study.

Justification for classification or non-classification

In a reproductive toxicity screen combined with repeated dose toxicity according to OECD 422 no fertility effects were seen and no developmental effects occured. Therefore, the substance is not classified for reproductive toxicity according to CLP (Regulation EC No 127272008) or DSD (Directive 67/548/EEC).