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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference Type:
Teratologic and postnatal evaluation of aniline hydrochloride in the Fischer 344 rat
Price CJ, Tyl RW, Marks TA, Paschke LL, Ledoux TA, Reel JR
Bibliographic source:
Toxicol. Appl. Pharmacol. 77, 465-478

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Exposure started GD 7
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Anilinium chloride
EC Number:
EC Name:
Anilinium chloride
Cas Number:
anilinium chloride
Constituent 2
Reference substance name:
aniline, hydrochloride
aniline, hydrochloride
Details on test material:
- Name of test material (as cited in study report): aniline hydrochloride
- Analytical purity: no data

Test animals

Fischer 344

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in sterile distilled water and the pH adjusted to 3 with 1 M HCl. Dosing solutions were stored in amber-coloured glass bottles at 5 °C, warmed to room temperature prior to daily dosing and used for dosing up to, but not exceeding, one week after formulation.

- Concentration in vehicle: 5, 15 or 50 mg aniline hydrochloride/mL
- Amount of vehicle (if gavage): 2 mL/kg
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
I. GD 7 to GD 20
II. day 7 of gestation through parturition
Frequency of treatment:
Duration of test:
up to post-natal day 60
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 10, 30 or 100 mg aniline HCl/kg bw/d

Doses / Concentrations:
0, 7, 22 or 72 mg aniline/kg bw/d
actual ingested
No. of animals per sex per dose:
21 - 24 animals /dose for the teratogenicity study; 12 - 15 dams/group for the evaluation of postnatal development
Control animals:
Details on study design:
- Dose selection rationale: Dose selection was based upon previous laboratory studies in the Fischer 344 rat and were expected to extend into the maternally toxic range.


Maternal examinations:
Dams were weighed on gestation day 0, prior to daily dosing and at termination and were observed for clinical signs of toxcicity at dosing and at termination. A minimum of 20 pregnant animals per treatment group were sacrificed on gestation day 20. Dams which had been treated with 10 or 30 mg/kg/d aniline HCl were killed by cervical dislocation. Dams which had been treated with vehicle, 100 mg/kg/d aniline HCl or hydroxyurea were killed under methoxyflurane anesthesia at 20 or 120 min after the final treatment and blood samples were collected by heart puncture which were then analyzed for methaemoglobin content (using a modification of the cyanmethemoglobin method (Kiese, 1974; Hegesh et al., 1970) and blood cell distribution (reticulocyte count, red blood cell count, white blood cell count, hematocrit, red blood cell size, red blood cell distribution width and platelet count). The following measures were recorded for each dam on gestation day 20: body weight, liver weight, spleen weight. Remaining dams were killed on postnatal day 30.
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 live and dead fetuses.
Fetal examinations:
From live festuses the following observations were recorded: uterine position, body weight, crown-rump length, placental weight, sex. Blood was also collected to determine methaemoglobin content and blood cell distribution (see above). Fetal liver and spleen weights were recorded.

- External examinations: Yes; all live fetuses
- Soft tissue examinations: Yes; half per litter
- Skeletal examinations: Yes; all per litter
- Head examinations: Yes; half per litter

On postnatal day 0, litter size and mortality were recorded as well as clinical signs of toxcicity for each live pup. Litters were then culled to a maximumlitter size of eight. Pup body weights were recorded on postnatal day 0, 2, 10, 25, 50 and 60. Pups were randomly selected for termination on postnatal day 0, 10, 25 and 50 to collect liver and spleen weights and blood samples (random selction of one pup per litter). All remaining pups were killed on postnatal day 60 and liver, spllen and testis weights were recorded. On postnatal day 30, randomly selected pups from each litter (two males and two females when possible) were evaluated for three min in an open field test (latency to leave the start square, numbers of squares traversed, grooms, rears, urination and defecation in the apparatus were recorded).
Heamatological data were analyzed by two tailed t test. For all other measures (except nominal scale), Kruskal-Wallis one-way analysis of variance by ranks and Mann-Whitney U test (two tailed) was used. Nominal level measurements were analyzes by the chi square test and/or Fisher's exact test (two tailed). The significance level for each statistical comparison was at least 0.05.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
7 other: mg aniline/kg bw/d
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
10 other: mg aniline/kg bw/d
Basis for effect level:
other: other:

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
Effect level:
72 other: mg aniline/kg bw/d
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

With the prenatal part of the study all treated dams survived until termination on gestation day 20. Signs of maternal toxicity were observed in terms of dose-related lower mean absolute body weight gain (weight gain gestation day 0 to 20 minus gravid uterine weight), which was statistically significantly decreased to 19.4 ± 1.5 g in the high-dose group (100 mg/kg bw/d) in comparison to that of the control (26.7 ± 1.5 g), as well as a dose-dependent statistically significant increase in mean relative spleen weight in the 10, 30 and 100 mg/kg bw dose groups amounting to 24.3, 25.5 and 36.7% body weight in comparison to that of the controls (19.2% of body weight). Dams of the high-dose (100 mg/kg bw/d) group revealed signs characteristic of aniline toxicity, e.g. (2-4-fold) increased methaemoglobin concentrations (up to 13.7%), significantly decreased red blood cell counts (in 9 dams) and significantly increased reticulocyte counts (in 8 dams). No differences were observed among vehicle controls and aniline treated groups with respect to pregnancy rates, number of corpora lutea per dam or number of implantation sites per dam. There were also no differences for the numbers of live fetuses per litter, average fetal body weight, crown-rump length or relative fetal spleen weight. Fetal examination exhibited a slight but statistically significantly increased relative liver weight of 8.0 ± 0.15% of body weight for the 100 mg/kg dose group in comparison to the control (7.7 ± 0.12% of body weight), whereas no such changes were observed at the 10 and 30 mg/kg bw dose levels. A low incidence of malformations was observed in all treatment groups: 3/178 (1.7%) fetuses in the vehicle control, and 0/181 (0%), 7/210 (3.3%), and 4/190 (2.1%) fetuses in the 10, 30, and 100 mg/kg bw dose groups. No statistically significant differences were observed between vehicle and aniline-treated litters with regard to the proportion of litters with one or more malformed fetuses, or in the percentage of malformed fetuses per litter. The evaluation of hematological parameters of the litters from the high-dose treated dams did not reveal any effects on percentage of reticulocytes, white or red blood cell counts, hematocrit or platelets, and on methaemoglobin concentrations in comparison to the vehicle controls. However, a slight but statistically significantly smaller red blood cell distribution width of 14.83 ± 0.16 versus 15.64 ± 0.14 in the control and increased red blood cell size (MCV) of 162.6 ± 0.55 μm3 versus 158.8 ± 1.15 μm3 in the controls was observed.

For the postnatal part of this investigation dams sacrificed at postnatal day 30 exhibited a significantly elevated relative spleen weight (0.238 ± 0.007 in 15 animals versus 0.215 ± 0.005 in the controls), elevated methaemoglobin concentrations (2.7 ± 0.3 in 16 animals versus 1.7 ± 0.2 in the controls) and an increased MCV (6 animals) in the high-dose group, whereas relative spleen weight was not increased in the 10 and 30 mg/kg/day treated groups. Litters born to aniline-treated dams did not differ from vehicle controls in live litter size, incidence of stillborn pups per litter, percentage of male pups per  litter, average (male and female) pup weight or average (male and female) pup length per litter, average relative spleen weight, or average relative liver weight on postnatal day 0. Pups of the 100 mg/kg/day derived group exhibited a statistically significant increase in red blood cell size (MCV) of 141.3 ± 1.7 μm3 versus 133.4 ± 0.72 μm3 in the controls. It is reported that no statistically significant differences were noted among vehicle control and aniline-treated groups for other hematological endpoints on postnatal day 0, or for any parameter in the hematological profile on postnatal day 10, 25, and 50 (data not provided). After culling on postnatal day 0 the remaining offspring was raised up to postnatal day 60. During this period incidental and transiently lower body weights as well as incidental and transiently higher relative spleen and relative liver weights in comparison to the controls were recorded. Male offspring of the 100 mg/kg/day derived group exhibited body weights below those of controls on postnatal day 0, 2, and 10, but not on postnatal day 25, 50 or 60; female offspring of the 100 mg/kg/day derived group exhibited body weights below those of controls on postnatal day 2, but not on postnatal day 0, 10, 25, 50 or 60. Also, a significant dose response trend for elevation of pup relative spleen weights with aniline treatment was observed on postnatal day 25, but not on postnatal day 10, 50, or 60. Statistically significantly different from controls, but not dose related higher pup relative liver weights were seen on postnatal day 25 in the 10  and 30 mg/kg/day derived groups and on postnatal day 50 in the 10 mg/kg/day derived group, but not on postnatal day 10 or 60. At termination on postnatal day 60, no differences among aniline-treated and vehicle control groups were found for male pup relative testis weight. When compared to the  controls for the progeny of treated dams there were indications of some pre-weaning mortality reported in terms of a higher number of total deaths (6, 15, and 13 pups in the 10, 30, and 100 mg/kg dose groups in comparison to 3 pups in the controls; litter size or total number of pups not  provided) and a slightly higher number of litters with one or more postnatal deaths (3/16, 4/15, and 5/16 in the 10, 30, and 100 mg/kg dose groups in comparison to 2/15 in the controls). The higher number of litters in which one or more postnatal deaths occurred (mostly before postnatal day 6 and not later than weaning) was not statistically significantly different in the aniline-treated groups in comparison to the controls. No deaths were observed after weaning up to postnatal day 60. The age of appearance for indices of physical development (pinna detachment, visible pilation, lower incisor eruption, eye opening, vaginal opening, and testis descent), behavioural development (surface righting, cliff avoidance, auditory startle, wire grasping, and mid-air  righting), or behaviour of offspring in an open field (postnatal day 30) were not affected by aniline treatment.

Applicant's summary and conclusion