2-naphthol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-naphthol

EC Number:

205-182-7

EC Name:

2-naphthol

Cas Number:

135-19-3

Molecular formula:

C10H8O

IUPAC Name:

2-naphthol

impurity 1

Reference substance name:

1-naphthol

EC Number:

201-969-4

EC Name:

1-naphthol

Cas Number:

90-15-3

Molecular formula:

C10H8O

IUPAC Name:

1-naphthol

Test material form:

solid: flakes

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Tsukuba Rearing Center
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: 5 weeks. Females: 10 weeks.
- Weight at study initiation: (P) Males: 162-174 g; Females: 239.5-269.4 g
- Housing: Individually in metal cage (220w × 270d × 190h mm) with a metal grid floor. From Day 14 of gestation to necropsy: in plastic reproduction cages for rats (350w × 400d × 180h mm) having a floor of paper pulp chips.
- Diet: CE-2 CLEA Japan diet. ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 + 1°C
- Humidity (%): 50 to 65%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12/12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The administration sample was prepared by adding an aqueous 0.5% sodium carboxymethyl cellulose solution as the vehicle to the test substance weighed out to the respective concentration, stirring the mixture to suspension, and adjusting the content such that the volume of liquid administered each time was 5 mL/kg body weight regardless of the dose.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 3 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Re-mating was not necessary

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Amount test substance was determined using a high-performance liquid chromatograph (analysis column: Inertsil ODS-2 (4.6 mm i.d. × 150 mm, particle diameter 5 µm, GL Science), mobile phase: water/acetonitrile (55.45), flow rate: 1.0 mL/min, column temperature: constant at approximately 40°C, determination wavelength: 275 nm, amount of sample introduced: 10 µL). As a result, the prepared samples having concentrations of 1 mg/mL and 50 mg/mL were confirmed to be stable for 8 days when stored in a refrigerator and protected from light.

Duration of treatment / exposure:

Males: 10 weeks prior to mating, during the mating period and until the day before necropsy (98 days).
Females: 2 weeks prior to mating, during mating (up to 3 weeks) and gestation and until day 20 of lactation (up to 78 days).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

25 rats/sex/group (to obtain 20 pregnant animals per group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a dose-range finding study, the high dose was set at 160 mg/kg, which is expected to result in toxic change with relatively high frequency.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males: once weekly (on administration days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 92) and on the necropsy day. Females: once weekly until copulation was confirmed (administration days 1, 8, 15, 22 and 29), after copulation was confirmed on gestation days 0, 7, 14, and 20, and after parturition on lactation days 0, 4, 7, 14, and 21. Of these, administration days 22 and 29 were excluded from the evaluation subjects because determinations were only for animals in which copulation was not confirmed.

FOOD CONSUMPTION:
- Males: determined for administration days 2-3, 9-10, 16-17, 23-24, 30-31, 37-38, 44-45, 51-52, 58-59, 65-66, 71-72, 79-80, and 86-87. Of these, administration days 79-80 and 86-87 were excluded from the subjects because rearing conditions differed with the animal during the mating period. Females: determined before mating for administration days 2-3 and days 9-10, after copulation was confirmed for gestation days 0-7, 7-14, and 14-20 and after parturition for lactation days 0-4, 4-7, 7-14, and 14-21.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Vaginal smear specimens from each animal were made and monitored beginning two weeks before administration started, once mating started and until copulation was confirmed, and each animal was classified as being in estrus, proestrus, or anestrus on the basis of cell structure.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: histopathology of male reproductive organs including assessment of spermatogenesis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, external abnormalities.

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed by exsanguination under pentobarbital sodium anaesthesia the day following the end of administration.
- Maternal animals: All surviving animals were sacrificed by exsanguination under pentobarbital sodium anaesthesia on Lactation Day 21.

GROSS NECROPSY
- Gross necropsy consisted of checking visceral organs for the presence of anomalies.

HISTOPATHOLOGY / ORGAN WEIGHTS
Reproductive organs were prepared for microscopic examination. Anomalous organs were fixed with 0.1 M phosphate buffer 10% formalin solution and stored.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected were sacrificed at Day 4 of 21 post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of checking for external abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
Anomalous organs were fixed with 0.1 M phosphate buffer 10% formalin solution and stored.

Statistics:

Fisher's exact probability was used to analyse the estrous cycle frequency by type, the copulation and fertility indices, and frequency of morphological anomalies in delivered pups. The histopathological findings were analysed by significant difference testing between the control and each NPO group using the Mann-Whitney U test for the graded data and the one-tailed Fisher's exact probability test for the positive grade total. Other data were tested for uniformity of variance of each group by the Bartlett method using the value obtained for each individual or the mean values for each litter as one sample. When variance was uniform, one-way layout variance analysis was performed and when there was significance between groups, multicomparison by Dunnett's method was performed. Meanwhile, when variance was zero or when variance was not uniform in any of the groups, Kruskal-Wallis analysis of ranks was performed, and when there was significance between groups, multicomparison was performed by Dunnett's method.

Reproductive indices:

Fertility index, delivery index, parturition index and copulation index.

Offspring viability indices:

Birth index, the live birth index and viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient salivation was observed in male rats exposed to the test substance, with the number of cases and frequency of salivation increasing with the concentration of substance administrated to the animals. In female animals, transient salivation was observed at 40 mg/kg bw/d.
Transient decreased locomotor activity was reported in male and female animals exposed to 40 and 160 mg/kg bw/d.
Transient nasal discharge was observed in female animals exposed to 40 mg/kg bw/d.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal in the 160 mg/kg group died on administration day 60. Other than the same transient decrease in locomotor activity after dosing and transient salivation after dosing that were observed among other animals of the same group, anomalies were not detected while this rat was alive. Necropsy revealed oesophageal perforation and signs of bleeding near the hilum of the lung and therefore, it was judged that this animal had died due to administration error. This animal was the subject of evaluations of morbid change attributed to administration error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight gain after parturition up to lactation day 4 was somewhat low in the 160 mg/kg group, but there was not a significant difference from the control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption on gestation days 0 through 7 in the groups dosed with 40 mg/kg or more and food consumption on lactations days 4 through 21 in the 160 mg/kg group were significantly low (p < 0.05, p < 0.01) when compared to the control. In addition, in the 10 mg/kg group, food consumption on lactation days 14 through 21 was significantly low (p < 0.05) when compared to the control. Weight gain was not significantly affected.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed hyperplasia of the forestomach squamous epithelium in the animals showing thickening of the mucosa of the forestomach.
No relevant histopathological changes were found in the pituitary glands, testes, epididymides, coagulating glands, seminal vesicles, prostates, ovaries, uterus, cervix and vagina and in the stomach of females.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Among the animals in which fertility was not confirmed, mild degeneration of the spermatocytes at the 14th stage in spermatogenesis focused on the seminiferous tubules was observed in one animal each of the 40 mg/kg and 160 mg/kg groups, and cell debris in the epididymis lumen was observed in animals of the 40 mg/kg group. Even among animals in which fertility had been confirmed, degeneration of spermatocytes at the 14th stage in spermatogenesis focused on the seminiferous tubules was observed in a few rats of each of the administration groups, and cellular debris was observed in the lumen of the epididymis in many of these animals. However, there was no significant difference in these findings between the control and each of the administration groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no significant difference in the frequency of animals whose estrous cycle changed after starting administration or the number of days until estrus recurred between the control group and each of the administration groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no significant difference between the control group and each of the administration groups in terms of the fertility index, the number of days required for copulation after pairing was started, and the number of times the estrus phase recurred during that time.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

2

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased locomotor activity observed at 160 mg/kg bw/d was concluded by the authors of the study to slightly inhibit maternal behaviour, resulting in a lower viability index in pups.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the 160 mg/kg group there was a slight increase in the offspring that died between the day of birth and day 4 after birth, however it was not statistically significant when compared to the control group. The authors of the study considered that this decreased viability index was related to the decreased locomotor activity in female animals exposed at 160 mg/kg bw/d, which impacted on their maternal behaviour.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly lower body weight on Day 21 pp was observed in female pups from animals exposed to 10 and 160 mg/kg bw/d. No statistically significant difference was observed in the 40 mg/kg bw/d group nor in male rats.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy of the pups revealed morphological changes such as deformities and variations in each group, including the control

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Exposure to 2-naphthol did not impact on the reproductive performance in male and female parental animals. A non-significant decrease of the viability index for pups from the highest-dose group was observed, considered by the authors of the study to be consecutive to the transient decreased locomotor activity observed in female P animals, which could have impacted on their maternal behaviour.

Executive summary:

The toxicity to reproduction of 2 - naphthol was investigated during a study conducted in accordance with the OECD Testing Guideline 415 (One- Generation Reproduction Toxicity Study). The study was GLP- compliant.

Groups of 25 male and female rats were exposed to 0, 10, 40 or 160 mg/kg bw/d of test substance. The highest- dose was selected based on the results of a dose- range finding study. Male animals received the test substance 10 weeks prior to mating, during the mating period and until the day before necropsy, while female animals were dosed 2 weeks prior to mating, during mating (up to 3 weeks) and gestation and until day 20 of lactation.

Parental animals were observed daily for general clinical signs. Bodyweight and food consumption were recorded on a weekly basis. Oestrous cycle was monitored beginning two weeks before administration started, once mating started and until confirmation of copulation. Following sacrifice of the animals, gross necropsy was conducted as well as microscopic examination of reproductive organs and abnormalities.

Viability, bodyweight and general status of pups were recorded. At necropsy, postmortem examinations were conducted.

Reproductive and offspring indices were calculated.

No parental animal died as a result of the treatment. Transient salivation and decreased locomotor activity were observed in parental animals from the 40 and 160 mg/kg bw/d groups. Although obvious inhibition of weight gain was not observed, decreased food consumption was observed during the early stages of gestation in the groups dosed with 40 mg/kg or more and beginning on day 4 of lactation in the 160 mg/kg group.

Exposure to the test substance did not impact on oestrous cycle and reproductive indices.

Necropsy revealed a thick region in the forestomach mucosa and the histopathology revealed mild hyperplasia of the forestomach mucosa squamous epithelium in some male animals dosed with 40 mg/kg or more. Necropsy of female rats did not reveal any anomalies related to the test substance.

2 - naphthol did not have observable effects on pups. There was no significant difference from the control in terms of viability.  However, in the 160 mg/kg group, a tendency toward a reduction in the birth index was observed and on day 4 there was a reduction in the viability index and a reduction in the number of live pups after litter size adjustment. This was considered by the authors of the study to be consecutive to the transient decreased locomotor activity observed in female parental animals, which could have impacted on their maternal behaviour.

The following values can therefore be derived from this study:

NOEL for male reproductive toxicity: 160 mg/kg bw/day (highest tested dose)

NOEL for female reproductive toxicity: 40 mg/kg bw/day

NOEL for toxicity to the offspring: 40 mg/kg bw/day