N-1-naphthylaniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available for the test article. In a 90d subchronic toxicity study, no adverse effects were observed in sexual organs.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The NOAEL for prenatal developmental toxicity was 150 mg/kg bw/d (highest dose tested).

The NOAEL for maternal toxicity was determined to be 50 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 141.2 - 192.8 g
- Housing: individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and BECKER & CO., Castrop-Rauxel, Germany (floor area about 800 cm²). Dust-free wooden bedding was used in this study. For enrichment, wooden gnawing blocks were offered (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet: ad libitum, Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The oily test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations the specific amount of test substance was weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer until it was completely dissolved (40°C for a maximum of about one hour). During administration (after the preparations had reached room temperature), the preparations were kept homogeneous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and the high-concentrations (15 and 150 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

From implantation to one day prior to the expected day of parturition (GD 6 to GD 19)

Frequency of treatment:

daily

Duration of test:

Until GD 20

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS
A check for dead animals was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20). A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

BODY WEIGHT
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION
Water consumption was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

HAEMATOLOGY
In the morning of GD 20 blood was taken from the retro-bulbar venous plexus from fasted animals. The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, heinz bodies

CLINICAL CHEMISTRY
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyltransferase, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

POST-MORTEM EXAMINATIONS
On GD 20, the dams were sacrificed. After the dams had been sacrificed, they were necropsied and assessed for gross pathology.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead fetuses

Fetal examinations:

At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

- Soft tissue examinations: The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

- Skeletal examinations: The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

- Water consumption, food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of post-implantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight were evaluated by simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings were evaluated by pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
- Proportions of fetuses with malformations, variations and/or unclassified observations in each litter were evaluated by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- For blood parameters with bidirectional changes a non-parametric one-way analysis was performed using the KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
Only pregnant dams were used for the calculations of mean maternal water consumption, food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. One female in group 0 (0 mg/kg bw/d), two females in group 1 (15 mg/kg bw/d) and three females in group 2 (50 mg/kg bw/d) were not pregnant.

Indices:

The conception rate (in %) = number of pregnant animals / number of fertilized animals x 100
The preimplantation loss (in %) = number of corpora lutea – number of implantations / number of corpora lutea x 100
The postimplantation loss (in %) = number of implantations – number of live fetuses / number of implantations x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some (20 out of 25) females of the high-dose group (150 mg/kg bw/d) and five females of the mid-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Salivation persisted in the respective animals only for some minutes after daily dosing (i.e. up to 10 minutes) and was initially observed on GD 8. This effect was assessed as treatment-related. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 15, 50 or 150 mg/kg bw/d during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of the test groups (0, 15, 50 or 150 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the dams in test group 3 (150 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. The body weight change of the high-dose dams was statistically significantly reduced on GD 6-8 (approx. 41% below control). If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the high-dose dams gained 9% or 8% less weight, respectively, in comparison to the concurrent control group. This effect was assessed as test substance- related. The mean body weights and the mean body weight gains of the low- and mid-dose dams (15 or 50 mg/kg bw/d) were generally comparable to the controls throughout the entire study period.

The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 3 (150 mg/kg bw/d - about 26% below the concurrent control value). Furthermore, the carcass weight of the high-dose dams was also statistically significantly reduced in comparison to the control group (about 5% below controls). These effects are assessed as test substance-related signs of maternal toxicity. The corrected body weight gain of test groups 1 and 2 (15 or 50 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, carcass weights remained also unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the dams in test group 3 (150 mg/kg bw/d) was statistically significantly reduced at the beginning of the treatment period (GD 6-13; up to 16% below control), but recovered afterwards. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the high-dose dams consumed 9% or 7% less food, respectively, in comparison to the concurrent control group. This effect was assessed as test substance-related. The mean food consumption of the dams in test groups 1 and 2 (15 and 50 mg/kg bw/d) was generally comparable to the concurrent control group throughout the whole study period.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

The mean water consumption of the dams in test group 3 (150 mg/kg bw/d) was statistically significantly increased from GD 10 onwards until scheduled sacrifice on GD 20. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the high-dose dams consumed 24% or 19% more water in comparison to the concurrent control group. This effect was assessed as test substance-related. The mean water consumption of the dams in test groups 1 and 2 (15 and 50 mg/kg bw/d) was generally comparable to the concurrent control group throughout the whole study period.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At gestation day 20 in dams of test group 3 (150 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values as well as mean corpuscular hemoglobin concentration (MCHC) were decreased, whereas relative reticulocyte counts were increased. Additionally, in dams of test group 3 (150 mg/kg bw/d) platelet counts were higher and relative eosinophil counts were lower compared to controls. Both means were within historical control ranges (platelets 844-1110 giga/L; relative eosinophils 0.8-1.7 %) and therefore these alterations were regarded as incidental and not treatment-related. No Heinz bodies were found in individuals of any test group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 20 in dams of test groups 1, 2 and 3 (15, 50 and 150 mg/kg bw/d) total bilirubin concentrations were higher compared to controls. The values in test group 1 were within the historical control range (bilirubin 0.95-1.85 μmol/L) those of test groups 2 and 3 were above this range. In test group 2 higher bilirubin levels were the only relevantly altered clinical pathology parameter. Therefore, the total bilirubin increase in test groups 1 and 2 were regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002). In dams of test group 3 (150 mg/kg bw/d) cholesterol levels were decreased. In dams of test group 3 (150 mg/kg bw/d) alanine aminotransferase (ALT) activities and urea levels were increased and total protein, albumin, globulin and creatinine levels were lower compared to controls. Urea levels were already increased in dams of test groups 1 and 2 (15 and 50 mg/kg bw/d). However, all changed values were within historical control ranges (ALT 0.68-1.20 μkat/L; urea 4.52-7.14 mmol/L; total protein 55.10-62.64 g/L; albumin 30.69-38.59 g/L; globulins 21.10-26.90 g/L; creatinine 22.8-29.3 μmol/L). Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test group 1-3 (15, 50 and 150 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings occurred at necropsy in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The conception rate reached 88% in the mid-dose group (50 mg/kg bw/d), 92% in the low-dose group (50 mg/kg bw/d), 96% in the control and 100% in the high-dose group (150 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study. There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 15, 50 and 150 mg/kg bw/d) with regard to conception rate, the mean number of corpora lutea and implantation sites or the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

haematology

water consumption and compound intake

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights were similar in all test groups (15, 50 and 150 mg/kg bw/d) and did not show any biologically relevant differences.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (15, 50 and 150 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus of test group 1 (15 mg/kg bw/d) had an external malformation (anal atresia). The total incidences of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in test groups 0 (severely malformed vertebral column in one female, misshapen turbositas deltoidea in one male), 2 (severely malformed skull bones in one male) and 3 (misshapen scapulae in two females) (0, 50 or 150 mg/kg bw/d). The total incidences of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data. For a better overview, all skeletal variations with statistically significant differences between control and the treated groups were compiled. All incidences were expressed on a fetus per litter basis and any statistically significant differences was determined. A few variations - affecting some elements of the vertebral column and ribs - were, at the top dose (150 mg/kg bw/d) and/or at the mid dose (50 mg/kg bw/d), present at statistically significantly incidences above the historical control range. An association with the treatment cannot be ruled out. The other findings were not considered to be treatment-related, because there is no dose-response.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two soft tissue variations were detected in test groups 0, 1 and 3 (0, 15 and 150 mg/kg bw/d), i.e. dilated renal pelvis and dilated ureter. The incidences of these variations were neither significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at higher incidences.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects, highest dose tested

Developmental effects observed:

no

Assessment of all fetal external, soft tissue and skeletal observations

Soft tissue malformations did not occur in any of the fetuses in this study. There were noted external and skeletal malformations in test groups 0, 1, 2 and 3 (0, 15, 50 and 150 mg/kg bw/d). The distribution of malformations about the groups was not related to the doses. Two fetuses were multiple-malformed. One female control group fetus had several skeletal malformations concerning the vertebral column and male mid-dose fetus (50 mg/kg bw/d) had several skeletal malformations concerning the skull. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of the test groups. Other malformations, such as anal atresia, misshapen scapula and misshapen tuberositas deltoidea, observed in test groups 0, 1 or 3, were scattered observations in individual fetuses of these test groups, are common for this rat strain and most of them can be found in the historical control data at comparable or higher frequency.

Total fetal malformations:

		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 246	22 229	22 219	25 262
Fetal incidence	N (%)	2 (0.8)	1 (0.4)	1 (0.5)	2 (0.8)
Litter incidence	N (%)	2 (8.3)	1 (4.5)	1 (4.5)	1 (4.0)
Affected fetuses/litter	Mean%	0.9	0.6	0.4	0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

External variations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for two soft tissue variations which were observed in fetuses of test groups 0, 1 and 3. A broad range of skeletal variations are equally distributed among the different test groups including control, if normal biological variation is taken into account, and can be found in the historical control data at similar frequencies. At 150 mg/kg bw/d altered rib cages (wavy ribs) were noted in 25 pups out of 13 litters. This resulted in a statistically significantly increased rate of 22.8% affected fetuses per litter. In addition incomplete ossification of the cervical arch (cartilage present) in 7 pups out of 3 litters (150 mg/kg bw/d) and 5 pups out of 4 litters (50 mg/kg bw/d) resulted in significantly increased rates of 5% or 3.7% affected fetuses per litter, respectively. The increase in the mid dose group is only slightly above the historical control range (0.0 - 3.2%).

Occurrence of statistically significantly increased skeletal variations (expressed as mean percentage of affected fetuses/litter):

Finding	Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d	Historical control data Mean % (range)
Supraoccipital hole(s)	0.0	2.7	2.2*	1.1	6.9 (0.0 – 30.1)
Incomplete ossification of cervical arch; cartilage present	0.0	0.8	3.7*	5.0*	0.3 (0.0 – 3.2)
Unossified sternebra; unchanged cartilage	1.7	4.1	6.6*	4.8	6.1 (0.0 – 20.7)
Wavy rib	8.0	5.2	1.7	22.8*	4.3 (0.0 – 8.7)

* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Those minor changes in connection with significant maternal toxicity in the high-dose group represent either minor departures from normal morphology not affecting morphology or slight delays of ossification. In the literature (Price CJ, Strong PL, Marr MC, Myers CB, Murray FJ. (1996); Nishimura M, Iizuka M, Iwaki S, Kast A. (1982); Hayasaka I, Tamaki F, Uchiyama K, Kato Z, Murakami K. (1985)) wavy ribs are described as a common reversible finding during postnatal development. As additionally the total incidence of skeletal variations is equally distributed among the different test groups including the control group, the single changes were considered to be a possible effect of treatment, but not as an adverse effect.

Total fetal variations:

		Test group 0 0 mg/kg bw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 150 mg/kg bw/d
Litter Fetuses	N N	24 246	22 229	22 219	25 262
Fetal incidence	N (%)	128 (52)	120 (52)	111 (51)	141 (54)
Litter incidence	N (%)	24 (100)	22 (100)	22 (100)	25 (100)
Affected fetuses/litter	Mean%	52.1	52.3	50.3	53.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Unclassified external and unclassified soft tissue observations did not occur in any fetuses of this study. A spontaneous origin was assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 15, 50 and 150 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no other effect of the compound on the respective morphological structures up to a dose of 150 mg/kg bw/d.

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused maternal toxicity (increased water consumption, reduced food consumption, decreased body weight gain and a regenerative anemia) in the high dose group (150 mg/kg bw/d). In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 50 mg/kg bw/d. Single variations - affecting some elements of the vertebral column and ribs - were, at the top dose (150 mg/kg bw/d) and/or at the mid dose (50 mg/kg bw/d), present at incidences outside the historical control range. Those minor changes in connection with significant maternal toxicity in the high dose group are assessed as a possible effect of the treatment, but not as adverse. The no observed effect level (NOEL) for prenatal developmental toxicity was therefore 15 mg/kg bw/d; the NOAEL was 150 mg/kg bw/d, the highest dose tested.

Executive summary:

The test item was examined for its prenatal developmental toxicity in pregnant Wistar rats. The test substance was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at dose levels of 0, 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group was dosed with the vehicle in parallel (corn oil). A standard dose volume of 4 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 22-25 females per group had implantation sites. Water consumption, food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The following test substance-related adverse effects/findings were noted:

Test group 3 (150 mg/kg bw/d):

Dams

• Increased mean water consumption from GD 10 onwards until scheduled sacrifice on GD 20. If calculated for the administration period (GD 6-19) the dams consumed 24% more water.

• Reduced food consumption on GD 6-13 (up to 16% below control). If calculated for the administration period (GD 6-19) the dams consumed 9% less food.

• Reduced body weight change on GD 6-8 (approx. 41% below control). If calculated for the administration period (GD 6-19) the dams gained 9% less weight.

• Distinctly lower corrected (net) body weight gain (about 26% below control). Furthermore, the carcass weight was also reduced (about 5% below controls).

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values as well as decreased mean corpuscular hemoglobin concentration (MCHC) in the dams.

• Decreased cholesterol levels in the dams.

• Increased relative reticulocyte counts and bilirubin levels in the dams.

Fetuses

• No test substance-related adverse effects in fetuses.

Test group 2 (50 mg/kg bw/d):

• No test substance-related adverse effects in dams, gestational parameters or fetuses.

Test group 1 (15 mg/kg bw/d):

• No test substance-related adverse effects in dams, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused maternal toxicity (increased water consumption, reduced food consumption, decreased body weight gain and a regenerative anemia) in the high dose group (150 mg/kg bw/d). In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 50 mg/kg bw/d. Single variations - affecting some elements of the vertebral column and ribs - were, at the top dose (150 mg/kg bw/d) and/or at the mid dose (50 mg/kg bw/d), present at incidences outside the historical control range. Those minor changes in connection with significant maternal toxicity in the high dose group are assessed as a possible effect of the treatment, but not as adverse. The no observed effect level (NOEL) for prenatal developmental toxicity was therefore 15 mg/kg bw/d; the NOAEL was 150 mg/kg bw/d, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 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

In a prenatal developmental toxicity study the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. The test substance caused neither mortality nor clinical symptoms of systemic toxicity in any of the exposed groups receiving 15, 50 or 150 mg/kg bw/d test material. Some females (20 out of 25) of the high-dose group (150 mg/kg bw/d) and five females of the mid-dose group (50 mg/kg bw/d) showed transient salivation after treatment. This salivation persisted in the respective animals for a few minutes immediately after each administration. It is considered to be treatment-related, likely resulting from bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The high dose of the test substance (150 mg/kg bw/d) caused significant increase in water consumption, decrease in food consumption (mainly at the beginning of treatment) and body weight gain as well as a distinct decrease in the corrected (net) body weight gain. These effects are considered to be treatment-related and adverse. No toxicologically relevant effect on water consumption, food consumption and body weight gain was noted for the animals exposed to 15 or 50 mg/kg bw/d 1-Naphthalinamine, N-phenyl-. Regarding clinical pathology in dams of test group 3 (150 mg/kg bw/d) a normocytic, hypochromic anemia was observed indicated by decreased red blood cell (RBC) counts, hemoglobin and hematocrit as well as mean corpuscular hemoglobin concentration (MCHS) values, but not changed mean corpuscular volume (MCV). The anemia was regenerative because of increased reticulocyte counts. An increased degradation of red blood cells can be assumed because of higher serum total bilirubin levels expressing an increased metabolism of free hemoglobin. Liver cell function was marginally affected because of decreased cholesterol levels in dams of test group 3. No differences of toxicological relevance were noted between the control and the treatment groups (15, 50 or 150 mg/kg bw/d) for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weights and sex distribution was noted at any dose level. Single variations - affecting some elements of the vertebral column and ribs - were, at the top dose (150 mg/kg bw/d) and/or at the mid dose (50 mg/kg bw/d), present at incidences outside the historical control range whereas the incidence of total fetal variations is comparable to the control group. Those minor changes in connection with significant maternal toxicity in the high dose group represent either minor departures from normal morphology not affecting morphology or slight delays of ossification. As there is also no evidence of an ontogenetic pattern in these single findings, the toxicological relevance is questionable. Thus, they were considered as possible effect of treatment, but not adverse. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose level.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused maternal toxicity (increased water consumption, reduced food consumption, decreased body weight gain and a regenerative anemia) in the high dose group (150 mg/kg bw/d). In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 50 mg/kg bw/d. Single variations - affecting some elements of the vertebral column and ribs - were, at the top dose (150 mg/kg bw/d) and/or at the mid dose (50 mg/kg bw/d), present at incidences outside the historical control range. Those minor changes in connection with significant maternal toxicity in the high dose group are assessed as a possible effect of the treatment, but not as adverse. The no observed effect level (NOEL) for prenatal developmental toxicity was therefore 15 mg/kg bw/d; the NOAEL was 150 mg/kg bw/d, the highest dose tested.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.