N-methylaniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

No specific studies are available for assessment of the possible effect of the test substance exposure on fertility. However, the repeated dose studies available (oral and inhalation), show no significant effects on reproductive organs of either male or female at concentrations that induce significant effects in the blood system and the target organ, the spleen. For aniline, a similar conclusion can be drawn. Therefore, no hazard is expected to be present for fertility at concentrations, which are at or below the NOEL(C) for repeated dose toxicity.

Effects on developmental toxicity

Description of key information

The NOAEL in pregnant females was established as 15 mg/kg bw/day.
This NOAEL value is based on no mortality of females, no changes in health condition status, no alteration to thyroid hormone measurements, no pathological findings in the dams and no dose-related changes in reproduction parameters.

The NOAEL for PRENATAL DEVELOPMENT was established as 15 mg/kg bw/day. This NOAEL is based on no altered growth and no significant treatment-related structural abnormalities in foetuses of pregnant rats. However, due to signs of toxicity observed at 40 mg/kg bw/day in pregnant females, the adverse effects observed on foetuses at the same dose level are considered as secondary to maternal toxicity.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

15 mg/kg bw/day

Additional information

A developmental toxicity study according to the OECD guideline 414 was performed on rats by oral administration of N-Methylaniline.

Before the start of experiments with laboratory animals the stability and homogeneity of application form were determined at the test facility. A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study.

After evaluating the results of the DRFE, doses 5, 15 and 40 mg/kg bw/day were selected for the main study. SPF quality male and female Wistar rats were used for testing. For the main study, after acclimatization the females were mated with males and the test item (at a dose of 5, 15 and 40 mg/kg bw/day) or vehicle (olive oil) control was administered daily to pregnant females from the 5th to the 19th day of pregnancy. The test item was administered in olive oil by stomach tube and adjusted to ensure the administered volume of 1 mL per 100 g of body weight for each of the dose groups. 

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study.

On the day of necropsy blood was collected from pregnant females to determine thyroid hormone levels (T3, T4, TSH). On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were assessed for changes on soft tissues and skeleton. The foetuses were weighed and the anogenital distances were measured. The thyroid glands were removed from the females and histological examination was performed.

There were no deaths of females during the study at any dose level. No adverse changes of health condition and no clinical symptoms of intoxication were observed in maternal animals following administration of the test item at any dose level.

The body weight in all dose levels were comparable to the control group. Probably toxicologically significant treatment-related effect on body weight increment and food consumption in maternal animals was observed. The body weight increment was statistically significantly decreased at the dose level 40 mg/kg bw/day. The corrected body weight of treated females was slightly decreased at the dose levels 15 and 40 mg/kg bw/day, with dose dependence and without statistical significance. Food consumption was statistically significantly decreased at all treated groups in comparison with dose dependence. The reduced food consumption was statistically significantly decreased at all treated groups in comparison with the control group from the 8th to the 20th day of pregnancy, on the 8th day of the pregnancy with dose dependence. The reduced food consumption is not adverse effect of the test item treatment at the dose levels 5 and 15 mg/kg bw/day, because the body weights and body weight increments in these dose levels were similar to the control group. Reduced food consumption without concomitant effect on body weight gain is likely a transient effect and probably not adverse (Ronald D. Hood: Developmental and Reproductive Toxicology: A Practical Approach, Third Edition).   

At the highest dose level, the reduction of food consumption correlate with the lower body weight increment. The changes in body weight increment and food consumption at the dose level 40 mg/kg bw/day were considered to be adverse.

Pathological examination of pregnant and non-pregnant females revealed pathological finding probably related to the test item treatment at the dose level 40 mg/kg bw/day. In this dose,
an enlarged and/or dark spleens were recorded in all females. Histological examination of spleen revealed congestion (mild to marked) in all 24 females at this dose level. Histological examination of spleens revealed congestion (mild to marked) in all 24 females at this dose level. Extramedullary hematopoiesis was found in 23 females and this finding is not a physiological condition. Drugs and chemicals that damage blood cells can cause intense extramedullary hematopoiesis.  Hemosiderosis of spleen was found in 23 females and ­this finding is associated with destruction of red blood cells of the spleen. The last finding was haemorrhage, which was found in 4 females at this dose level. The observed pathological changes are multiple, significant, consistent and related to the treatment of the test item.

Evaluation of uterine weights (absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects.

Macroscopic structure of examined organs of pregnant females and reproduction parameters (number of females with live foetuses and number of live foetuses), were unaffected by treatment with the test item. Number of resorptions were slightly increased at the dose level 40 mg/kg bw/day, without statistical significance and dose dependence. One female at the same dose level became pregnant and then all implanted conceptuses in uterus were totally resorbed. Post implantation losses (IUDL) were increased at the dose level 40 mg/kg bw/day, without statistical significance and dose dependence. These findings in reproductive parameters (resorption, IUDL) were caused probably by the test item treatment.

Examination of the thyroid glands (absolute and relative weight of thyroid gland, histological examination of thyroid gland) was performed. Serum level of thyroid hormone T4 was statistically insignificantly increased at the dose level 15 mg/kg bw/day, without dose dependence. The concentration of TSH was decreased at the dose level 5 mg/kg bw/day and slightly increased at the dose level 40 mg/kg bw/day, both without dose dependence and statistical significance.

Histological examination of thyroid glands revealed mild atrophy, mild degeneration, hyperplasia C cells and/or mild hypertrophy C cells. The found changes are isolated, weakly expressed and do not deviate from the natural viability of the thyroid histological picture of clinically healthy animals. These findings were also found at the control group and are not related to the treatment of the test item.

During necropsy was reported foetal mortality of five foetuses at the dose level
40 mg/kg/ bw/day.

The mean value of body foetal weight was decreased at the dose level 40 mg/kg bw/day, with statistical significance in male foetuses and without dose dependence. This reduction of foetal body weight is associated to the decrease of body weight increments of pregnant females at the same dose level. The statistically significantly increased foetal body weight was observed in all foetuses and female foetuses at the dose level 5 mg/kg bw/day. These increases were without biological and toxicological significance.

The mean AGD of male foetuses was slightly decreased at the dose level 40 mg/kg bw/day with statistical significance. This reduction is related to the decreased body weight of male foetuses at the same dose level, because corrected AGD at this dose level was similar to the control group. Other mean AGD and corrected AGD of male and female foetuses at other dose levels were comparable to the control group.

 Detailed necropsy of foetuses did not reveal an increase of external and visceral variations and malformations at any dose level. Only five dead foetuses were found at the dose level 40 mg/kg bw/day. One foetus was found without tail at the dose level 5 mg/kg bw/day.

There were statistically significant differences without toxicological significance in proportion in number of foetuses and litters during the statistical evaluation of skeletal findings.

The incidence of litters with incomplete ossification of parietal bone was increased at the dose level 40 mg/kg bw/day (80.00 % – 86.96 % – 79.17 % – 100.00 %). The increased proportions of litters with incomplete ossification of basisphenoid (15.00 % – 8.70 % – 25.00 % – 30.00 %) were recorded at the dose levels 15 and 40 mg/kg bw/day. These increases are without statistical significance and dose dependence.

The proportions of litters with bipartite ossification of ossification sites of sternebra were statistically insignificantly increased at the dose levels 15 and 40 mg/kg bw/day (5.00 % – 8.70 % – 12.50 % – 30.00 %), with dose dependence. The increased proportions of litters with asymmetric bipartite ossification of vertebrae thoracic centrum (5.00 % – 13.04 % – 25.00 % – 5.00 %) were recorded at the dose levels 5 and 15 mg/kg bw/day. The incidence of litters with dumbell ossification of vertebrae thoracic centrum (70.00 % – 65.22 % – 95.83 % – 75.00 %) and with asymmetric dumbbell ossification of vertebrae thoracic centrum (40.00 % – 43.48 % – 62.50 % – 50.00 %) were increased at the dose level 15 mg/kg bw/day. These increases are without statistical significance and dose dependence. Changes in the size, shape, or symmetry of sternebrae or vertebral centra are transient and have no implications for the health or survival of the offsprings.

Two supporting studies are available on two similar substances, which also represent the two main impurities of the test item.

In the first study carried out on aniline, timed-pregnant Fischer 344 rats were dosed by gavage with aniline hydrochloride (10, 30, or 100 mg/kg/day), a positive control agent (hydroxyurea, 200 mg/kg/day), or vehicle (distilled water) on gestational days (gd) 7 through 20 or gd 7 through parturition. At termination on gd 20 confirmed-pregnant dams exhibited characteristic signs of aniline HCl toxicity, i.e., methemoglobinemia, increased relative spleen weight, decreased red blood cell (RBC) count, and hematological changes indicative of increased hematopoietic activity. High-dose dams exhibited mild methemoglobinemia, increased relative spleen weight, and increased RBC size at termination on postnatal day (pnd) 30. At termination on gd 20, fetuses from aniline-treated dams exhibited increased relative liver weight and enhanced hematopoietic activity, but no evidence of an embryolethal or teratogenic effect was observed. Postnatal signs of toxicity in litters from aniline-treated dams (i.e., decreased body weight, elevated relative liver weight, and elevated relative spleen weight) were transient, and no evidence of toxicity was observed in pups surviving to pnd 60. Hydroxyurea (200 mg/kg/day) administered by gavage proved to be an excellent positive control for embryotoxicity, maternal toxicity, teratogenicity, and postnatal maturational deficits in the Fischer 344 rat. In conclusion, aniline hydrochloride was not teratogenic to Fischer 344 rats, even at maternally toxic doses: transient signs of toxicity were observed postnatally in the offspring in conjunction with mild, but persistent signs of maternal toxicity through pnd 30.

In the second supporting study carried out on N,N-dimethylaniline, the no effect levels for embryonal and fetal development of rats exposed in utero to aniline and p-nitroaniline are below concentrations of 560 and 1200 mg/kg bw/d, respectively. The NOEL of N,N-dimethylaniline for teratogenic effects in rats was at 365 mg/kg bw/d. 

Data obtained in these two studies, confirm a general toxicity characterising this class of substances.

 

Justification for classification or non-classification

According to the CLP Regulation n.1272/2008, Table 3.7.1 (a) "Hazard categories for reprodictive toxicant", a substance should be allocated in Category 2 " Suspected human reproductive", "when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.

Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects."

In the study carried out on the test item, significant maternal toxicity was observed at the highest dose tested (i.e. 40 mg/kg bw). Statistically significant decreased body weight increment, reduced food consumption and marked adverse effects on spleen were observed at this dose level.

At the same dose level 5 foetuses were found dead. Furthermore, mean value of body foetal weight was decreased and a slightly decreased mean AGD was also observed. 

However, the adverse effects observed on foetuses at 40 mg/kg bw were considered as secondary to maternal toxicity.

The test item was therefore not classified for developmental toxicity according to the criteria set out in the CLP Regulation.