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Toxicity to reproduction

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one-generation reproductive toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 1993 - 4 May 1995
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD-Guideline Study conducted under GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BNE
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.8 %
- Lot/batch No.: 21215
- Expiration date of the lot/batch: December 31, 1994
- Stability under test conditions: up to 3 weeks
- Storage condition of test material: At room temperature in the dark

Test animals

Details on test animals or test system and environmental conditions:
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, CH 4414 Füllinsdorf / Switzerland
- Age at study initiation: (P) males 6 wks, females 9 wks
- Weight at study initiation: (P) Males: 132.8 - 159.8 g; Females: 160.4 - 189.4 g
- Housing: individually

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

- Temperature (°C): 22 +- 3 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12 h

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
The test article was dissolved in acetone, mixed with granulated feed in a Buehler Mixer, type DDMA-0.5 and pelleted in a Buehler pelleting machine type DFPL. Water was added to each feed preparation at an approximate 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for approximately 48 hours before storage.

- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): Kliba 343 (batches 86/93 and 70/94 rat/mouse maintenance diet, Klingentalmuehle AG, CH 4303 Kaiseraugst, Switzerland)
- Storage temperature of food: room temperature

- Justification for use and choice of vehicle (if other than water): solubility reasons
- Concentration in vehicle: no data
Details on mating procedure:
- M/F ratio per cage: 1.0
- Length of cohabitation: up to 16 days
- Proof of pregnancy: sperm in vaginal smear / vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analysis of the test article, its content, homogeneity and stability in the feed pellets were determined before starting the test. In this study, feed samples for conten and homogeneity were taken before the start of the prepairing period and at the end of gestation/start of lactation period. The analyses were performed in the analytical laboratories of RCC Umweltchemie AG according to an analytical method supplied by the sponsor.
Duration of treatment / exposure:
the P generation animals of both sexes received the test article in the diet prior to pairing (males for 70 days, females for 14 days). The animals continued to receive the diets during pairing until sacrifice. The dams were sacrificed on day 21 post partum, the males were sacrificed on days 29 or 30 of the after pairing period.
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 600, 3000, 15000 ppm
nominal in diet
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were proposed by the sponsor based upon the results of a previous study.
- Rationale for animal assignment (if not random): random
- Other:
Positive control:


Parental animals: Observations and examinations:
- Time schedule: twice per day
- Cage side observations checked in table [No.?] were included.

- Time schedule: once or twice per day

- Time schedule for examinations: weekly

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
During the treatment period prior to pairing, vaginal smears were taken from all females to determine estrous cycles. The various stages of the estrous cycle (proestrus, estrus, metestrus, diestrus) were recorded.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
prostate, seminal vesicales with coagulating gland, testes with epididymides and pituitary were examined histopathological
Litter observations:
Pups were weighed individually on days 0 (if possible) and/or 1, 4, 7, 14, 21 of the lactation period. To prevent cannibalism immediately after birth the pups were weighed individually on day 0 post partum but not tattooed. The dams and pups were observed daily for survival and behavioral abnormalities in nesting and nursing.
Postmortem examinations (parental animals):
- Male animals: All surviving animals were sacrificed on days 29 or 30 of the after peiring period.
- Maternal animals: All surviving animals were sacrificed on day 21 post partum.

- Gross necropsy consisted of macroscopical examination for any structural abnormalities or pathological changes.

Any gross lesions, the ovaries, pituitary, prostate, seminal vesicles with coagulating gland, testes with epididymides, uterus and cervix and vagina were prepared for microscopic examination.
Postmortem examinations (offspring):
external and internal examination for abnormalities.
The following statistical methods were used to analyse body weights, food consumption and reproduction data:
- Means and standard deviation of various data were calculated.
- Univariate one-way analysis of variance was used to assess significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Birth Index
Offspring viability indices:
Viability index, weaning index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

In the high dose female group, the mean daily food consumption and mean body weight were reduced during the prepairing, gestation and lactation periods. this finding was considered to be test article- related.
In the mid-dose female group, very slight toxic effects were characterized by minimal reductions of mean daily food consumption during gestation and lactation, and slightly reduced body weight gain during prepairing, gestation and lactation.
The mean daily food consumption and mean body weights of low-dose gemale group were similar to those of control females throughout the prepairing gestation and lactation periods.

No test article-related effects upon mean daily food consumption and mean body weights of parent males were evident.

In the high-dose female group, the following test article-related findings were noted:
- reduced mean number of implantations per dam,
- increased post-implantation loss
- reduced birth index
- reduced mean litter size.

These parameters were unaffected in groups 2 and 3.

The following parental reproduction data were considered to be unaffected by the treatment with the test article at all dose levels:
- mating performance and fertility
- mean precoital time
- mean duration of estrous cycle
- mean duration of gestation
- mean post-natal loss
- mean breeding loss.

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
ca. 600 mg/kg diet
Based on:
test mat.
Basis for effect level:
other: overall effects body weight; food consumption and compound intake
Dose descriptor:
Effect level:
ca. 3 000 mg/kg diet
Based on:
test mat.
Basis for effect level:
other: number of implantation sites; birth index; litter size;

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

In male and female offspring of the mid- and high-dose group, the mean bith weight (day 0 post partum) and body weight development (days 14-21 post partum) was generally reduced when compared with that of the control offspring.
These findings were considered to be related to the treatment with the test article.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

On the basis of this one-generation reproduction study with 2-(phenylmethoxy)naphthalene in Wistar rats, the no-observable adverse effect level was 600 ppm (mg/kg diet) for the maternal organism, 3000 ppm for maternal reproduction parameters and neonatal organism.
There was no evidence for any teratogenic activity of the test article.
Executive summary:

The purpose of this one-generation reproduction toxicity study was to provide general information concerning the effects of the test article on reproductive functions as assessed by gonadal function, estrous cycle, mating behaviour, conception, parturition, lactation, weaning and the growth and development of the offspring. The study also provided information about the effects of the test article on neonatal morbidity, mortality, behavior and preliminary data on teratogenesis.

The control group received untreated diet. Each group of the P generation consisted of 25 male and 25 female rats. The animals received the following nominal dietary concentrations:

Group 1: 0 ppm (mg/kg diet, control)

Group 1: 600 ppm (mg/kg diet)

Group 1: 3000 ppm (mg/kg diet)

Group 1: 15000 ppm (mg/kg diet)

The following results were obtained:

Parental Data: P Generation

General Tolerability

All parent animals survived to scheduled necropsy and no test article-related clinical symptoms were evident.

The food consumption and body weight gain of parent males was unaffected by treatment.

Reductions of food consumption were noted throughout the study in females treated at 15000 ppm, and during gestation and lactation in females treated at 3000 ppm. Body weight gain was reduced throughout the stury in females treated at dose levels of 300 and 15000 ppm. these findings were considered to be test article related.

The food consumption and body weight gain of females treated at 600 ppm showed no test article-induced differences.

Reproduction data

Parent females treated with 15000 ppm showed reduced mean number of implantations per dam, increased post-implantation loss, reduced birth index and reduced mean litter size. No test article-related effects were evident in females treated with 3000 and 600 ppm.

The remaining reproduction parameters (mating performance and fertility, duration of estrous cycles, mean precoital time, duration of gestation, postnatal and breeding losses) were unaffected at all dose levels.


All abnormal macroscopical findings were considered to be incidental. There was no histopathological evidence of toxicity.

Litter Data: F1 Pups

At 15000 and 3000 ppm, reduced mean birth weight were noted in pups on day 0 post partum. Although generally similar in these groups on days 4 and 7 post partum, reduced body weight gain was ascertained in these pups from days 14 -21 post partum.

No evidence of test article-related effects on birth weights or body weight gain were seen in pups at 600 ppm.

The sex ratios of all groups compared favorably and no test article-related abnormal findings were ascertained during first litter check, during lactation or during necropsy.