N,N-dimethyl-p-toluidine

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Sub-chronic toxicity study with inclusion of reproductive endpoints

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Materials and methods

Principles of method if other than guideline:

The test chemical was given by oral gavage to rats for 14 weeks in order to evaluate the toxicological effects on the substance on several endpoints including reproductive endpoints such as sperm parameters, estrous cyclicity, and histology of reproductive organ tissues.

GLP compliance:

yes

Remarks:

The study was performed according to the Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58).

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-p-toluidine

EC Number:

202-805-4

EC Name:

N,N-dimethyl-p-toluidine

Cas Number:

99-97-8

Molecular formula:

C9H13N

IUPAC Name:

N,N,4-trimethylaniline

Test material form:

liquid

Details on test material:

- Name of test material: Benzenamine, N,N,N-trimethyl-
- Common Name: N,N-Dimethyl-p-toluidine
- Molecular formula: C9H13N
- Molecular weight: 135.209 g/mol
- Smiles notation: c1(N(C)C)ccc(C)cc1
- InChl: 1S/C9H13N/c1-8-4-6-9(7-5-8)10(2)3/h4-7H,1-3H3
- Substance type: Organic
- Physical state: liquid
- Purity: Greater than 99%.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: Rats were 5 to 6 weeks old at the beginning of the studies.
- Weight at study initiation:
- Fasting period before study:
- Housing: Male rats were housed up to 5 per cage
1. Cages : Polycarbonate (Lab Products, Inc., Seaford, DE), changed weekly (male mice) or twice weekly
2. Bedding: Irradiated Sani-Chips (P.J. Murphy Forest Products Corp., Montville, NY), changed weekly (male mice) or twice weekly
3. Cage Filters: Spun-bonded polyester (Snow Filtration Co., Cincinnati, OH), changed every 2 weeks
4. Racks: Stainless steel (Lab Products, Seaford, DE), changed and rotated every 2 weeks
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period:Animals were quarantined for 11 (male rats), 12 (female rats),

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: October 20 (males) or 21 (females) 2004 to January 19 (males) or 20 (females) 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared by adding the appropriate amount of the test chemical to corn oil to achieve the desired concentration. Dose formulations were prepared three times for the 3-month studies. The 400 mg/mL dose formulation was prepared and observed to be a true solution, therefore, no homogeneity or gavageability studies were performed. Stability studies of a 1.0 mg/mL formulation in corn oil were performed using GC/FID. Stability was confirmed for up to 44 days for formulations stored in amber glass containers sealed with Teflon®-lined lids, protected from light, at up to room temperature and for at least 3 hours under simulated animal room conditions. Formulations were administered at a volume of 2.5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the 3-month study, the dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed and used, all 13 for rats were within 10% of the target concentrations; all 13 of the animal room
samples for rats were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

Core study: 5 days/week for 14 weeks
Clinical pathology study: 5 days per week for a total of 25 days.

Details on study schedule:

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 62.5, 125, 250, 500 and 1000 mg/kg bw (5 times per week) for a total of 14 weeks (core study rats). Additional groups (10 rats/sex/dose) treated as described above, but for 25 days, were also included in the study protocol (clinical pathology study).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg bw (5 days/week) (nominal)

Remarks:

Vehicle alone (corn oil)

Doses / concentrations 2

Dose / conc.:

62.5 other: mg/kg bw (5 days/week) (nominal)

Doses / concentrations 3

Dose / conc.:

125 other: mg/kg bw (5 days/week) (nominal)

Doses / concentrations 4

Dose / conc.:

250 other: mg/kg bw (5 days/week) (nominal)

Doses / concentrations 5

Dose / conc.:

500 other: mg/kg bw (5 days/week) (nominal)

Doses / concentrations 6

Dose / conc.:

1 000 other: mg/kg bw (5 days/week) (nominal)

No. of animals per sex per dose:

Core study:
10 rats per sex per dose

Clinical pathology study:
10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The acute oral LD50 value in rats was 1,650 mg/kg.The doses for the 3-month studies were selected based on these LD50 values to deliver 0, 62.5, 125, 250, 500, and 1,000 mg/kg in rats.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.

Examinations

Parental animals: Observations and examinations:

The animals were observed twice day and weighed once per week. Blood samples were collected on day 25 (clinical pathology rats) and on day 88 (core study rats). Haematological parameters included: haematocrit, haemoglobin, methaemoglobin concentrations, erythrocytes, reticulocytes, nucleated erythrocytes, platelets, Heinz body counts, MCV, mean cell haemoglobin, mean cell haemoglobin concentrations, leukocyte count, and leukocyte differentials. Clinical chemistry parameters included
urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatinine kinase, sorbitol dehydrogenase, and bile acids.

Oestrous cyclicity (parental animals):

At the end of the 3-month period, vaginal cytology evaluations on core study rats administered 0, 62.5, 125, or 250 mg/kg. For 12 consecutive days prior to schedule terminal kill, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squa-mous epithelial cells were determined and used to ascer-tain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus).

Sperm parameters (parental animals):

Spermatid and sperm samples were collected from male rats treated at 0, 62.5, 125 and 250 mg/kg, with the following parameters investigated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis.

Postmortem examinations (parental animals):

All core study animals were necropsied, and the following organs were weighed: heart, right kidney, live, lung, right testis, and thymus. In addition to lesions, the following organs were examined histologically to a no-effect-level: adrenal gland, bone (including marrow), brain, clitoral gland, oesophagus, heart (including aorta), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung (and mainstem bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen,stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Various statistical methods were used to determine treatment-related effects on survival, histology, continuous variables. Historical control data was also taken into consideration during the data interpretation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity were observed at ≥250 mg/kg and included cyanosis, abnormal breathing, and lethargy.

Mortality:

mortality observed, treatment-related

Description (incidence):

All rats treated at 1000 mg/kg died by study day 3. With the exception of one male rat treated at 500 mg/kg, all rats treated at ≤500 mg/kg survived to planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in terminal body weight were observed at ≥62.5 mg/kg. Male rats treated at 62.5, 125, and 250 mg/kg weighed approx. 9.5, 11.6, and 22.9% less, respectively, compared to the control group by the end of the study period. The corresponding decreases for female rats treated at 62.5, 125, and 250 mg/kg were 5.2, 10.8, and 9.8%, respectively.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Methemoglobinemia and effects secondary to methemoglobinemia were observed at ≥62.5 mg/kg in both genders. The hematology findings were consistent with methemoglobinemia and Heinz body formation resulting in a macrocytic, hypochromic, responsive anemia. In general, these changes were dose-related, occurred at both timepoints evaluated, and involved all dosed groups of both sexes. The methemoglobinemia was described by a considerable treatment-related increase in methemoglobin values. The anemia was characterized by dose-related decreases in the erythron including decreases in hematocrit values,
hemoglobin concentrations, and erythrocyte counts. The greatest magnitudes of decrease occurred in the 500 mg/kg groups on day 25; the decrease was greater than 20% for hematocrit and hemoglobin values and close to 40% for erythrocyte counts. By week 14, there was some amelioration in the severities of the anemia. Erythrocyte macrocytosis was characterized by increases in mean cell volume and mean cell hemoglobin values indicating that the circulating erythrocytes were larger than those of the concurrent vehicle controls. Erythrocyte hypochromia was evidenced by decreases in mean cell hemoglobin concentration values, indicating that the circulating erythrocytes did not have the normal intracellular hemoglobin content. An erythropoietic response to the anemia was characterized by substantially increased reticulocyte and nucleated erythrocyte counts. Decreases in leukocyte counts occurred in 250 and 500 mg/kg male and female rats on
day 25. Decreases in lymphocyte counts mimicked the leukocyte count decreases; these changes were consistent with physiologic responses to stress.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On day 25, markers of hepatocellular injury, serum activities of alanine aminotransferase and sorbitol dehydrogenase, demonstrated dose-related increases in essentially all dosed groups of males and females; the 500 mg/kg animals had increases of greater than threefold. By week 14, the increases in alanine aminotransferase and sorbitol dehydrogenase activities had ameliorated or resolved in all dosed groups. Serum concentrations of total bile acids, a marker of
hepatic function/injury and cholestasis, were increased in higher-dose animals; the 500 mg/kg groups were the most consistently affected demonstrating a greater than threefold increase at both timepoints. Another marker of cholestasis (alkaline phosphatase activity), however, demonstrated decreases (day 25) or no change (week 14). Thus, it would appear that the increases in bile acid concentrations were not related to a cholestatic event. Serum albumin concentrations (and by extension, serum total protein) were increased in essentially all dosed male and female groups at both timepoints. The increases in albumin and total protein were proportional, suggesting that the increases were related to a physiologic hemoconcentration-type response (i.e.,dehydration). This supposition was supported by the substantially lower body weights suggesting that the treated animals did not eat and, therefore, drink as expected.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological findings included pigmentation of the liver at ≥62.5 mg/kg; hepatocyte hypertrophy at ≥125 mg/kg; hepatocyte necrosis at ≥62.5 mg/kg; degeneration of olfactory epithelium at ≥62.5 mg/kg; hyperplasia and metaplasia of the respiratory epithelium of the nose at ≥125 mg/kg; capsule fibrosis, congestion, hypertrophy, lymphoid follicle atrophy of the spleen at ≥125 mg/kg; nephropathy, papillary necrosis and mineralisation of the kidneys at ≥125 mg/kg.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Oestrous cycle lengths were significantly increased at 125 mg/kg (mean, 5.56 days) and 250 mg/kg (mean 5.38 days) compared to the control group (mean 4.95). The numbers of cycling female rats at 0, 62.5, 125 or 250 mg/kg were 10, 10, 9 and 4, respectively. The numbers of females with regular oestrous cycle at 0, 62.5, 125 or 250 mg/kg were 9/10, 10/10, 8/9, and 4/4, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No significant effects on spermatid or epididymal spermatozoal measurements were observed at any examined dose level (i.e. at 0, 62.5, 125 or 250 mg/kg). Decreased epididymis and testis weights were observed in male rats treated at 250 mg/kg; however no histopathological effects attributed to the test chemical
were reported in testis (with epididymis and seminal vesicle).

Reproductive performance:

not examined

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic toxicity

Effect level:

62.5 other: mg/kg bw (5 days/week) (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Sperm parameters

Effect level:

250 other: mg/kg bw (5 days/week) (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

3

Key result

Dose descriptor:

NOAEL

Remarks:

Estrous cyclicity

Effect level:

62.5 other: mg/kg bw (5 days/week) (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

4

Key result

Dose descriptor:

LOAEL

Remarks:

Estrous cyclicity

Effect level:

125 other: mg/kg bw (5 days/week) (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Results: F1 generation

Effect levels (F1)

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

not measured/tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

125 other: mg/kg bw (5 days/week)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Any other information on results incl. tables

TABLE 1.Survival and Body Weight of Rats in the 3-Month Gavage Study ofN,N-Dimethyl-p-toluidine^a

 

Dose (mg/Kg)	Survival^b	Initial Body Weight (g)	Final Body Wight (g)	Change in Body Weight (g)	Final Weight Relative To Controls (%)
Male
0	10/10	99 ± 3	327 ± 2	228 ± 4
62.5	10/10	99 ± 3	296 ± 5**	197 ± 5**	90
125	10/10	98 ± 3	289 ± 8**	191 ± 8**	88
250	10/10	97 ± 2	252 ± 5**	155 ± 4**	77
500	9/10^c	99 ± 3	234 ± 9**	135 ± 8**	72
1,000	0/10^c	99 ± 3	—	—	—
Female
0	10/10	93 ± 3	193 ± 3	100 ± 2
62.5	10/10	92 ± 3	183 ± 3*	91 ± 2*	95
125	10/10	92 ± 3	172 ± 4**	80 ± 2**	89
250	10/10	93 ± 3	174 ± 3**	80 ± 3**	90
500	10/10	92 ± 2	175 ± 3**	82 ± 2**	91
1,000	0/10^c	92 ± 2	—	—	—

 

* Significantly different (P≤0.05) from the vehicle control group by Williams’ test

** P≤0.01

 ^a  Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals 

     surviving to the end of the study.

 ^b   Number of animals surviving at 14 weeks/number initially in group

^c    Week of deaths: 1

 

 

TABLE 2.Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Gavage Study ofN,N-Dimethyl-p-toluidine^a

	Vehicle control	62.5 mg/Kg	125 mg/Kg	250 mg/Kg
n	10	10	10	10
Weights (g)
Necropsy body wt	327 ± 2	296 ± 5**	289 ± 8**	252 ± 5**
L. Cauda epididymis	0.1440 ± 0.0033	0.1259 ± 0.0046*	0.1392 ± 0.0050	0.1178 ± 0.0049**
L. Epididymis	0.4241 ± 0.0049	0.3955 ± 0.0116	0.4170 ± 0.0102	0.3740 ± 0.0101**
L. Testis	1.4584 ± 0.0189	1.5586 ± 0.0776	1.4644 ± 0.0305	1.4121 ± 0.0487
Spermatid measurements
Spermatid heads (10^6/testis)	172.00 ± 4.11	172.00 ± 5.31	180.00 ± 5.53	167.13 ± 6.96
Spermatid heads (10^6/g testis)	129.6 ± 3.0	124.4 ± 6.7	135.0 ± 4.6	131.5 ± 4.2
Epididymal spermatozoal measurements
Sperm motility (%)	82.2 ± 1.2	81.2 ± 1.2	82.8 ± 0.9	81.3 ± 0.8
Sperm (10^6/cauda epididymis)	98.63 ± 9.33	81.88 ± 4.89	82.13 ± 3.57	73.13 ± 5.81
Sperm (10^6/g cauda epididymis)	682 ± 58	659 ± 47	598 ± 33	617 ± 32

 

* Significantly different (P≤0.05) from the vehicle control group by Dunnett’s test

 ** Significantly different (P≤0.01) from the vehicle control group by Dunnett’s test (cauda epididymis weight) or Williams’

    test (body and epididymis weights)

 ^aData are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett’s     

   test (testis weight) or Dunn’s test (spermatid and epididymal spermatozoal measurements)

 

Table3.Estrous Cycle Characterization for Female Rats in the 3-Month Gavage Study ofN,N-Dimethyl-p-toluidine^a

	Vehicle	62.5 mg/Kg	125 mg/Kg	250 mg/Kg
Number weighed at necropsy	10	10	10	10
Necropsy body wt (g)	193 ± 3	183 ± 3*	172 ± 4**	174 ± 3**
Proportion of regular cycling females^b	9/10	10/10	8/9	4/4
Estrous cycle length (days)	4.95 ± 0.17	5.10 ± 0.15	5.56 ± 0.32^c	5.38 ± 0.47^d
Estrous stages^e(% of cycle)
Diestrus	51.7	55.8	59.2	60.0
Proestrus	12.5	13.3	12.5	7.5
Estrus	25.0	23.3	17.5	13.3
Metestrus	4.2	6.7	8.3	7.5
Not clear or no cells observed	6.7	0.8	2.5	11.7

 

* Significantly different (P≤0.05) from the vehicle control group by Williams’ test

 ** P≤0.01

^aNecropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunn’s test (estrous cycle length).

^b Number of females with a regular cycle/number of females cycling

^c Estrous cycle was longer than 12 days or unclear in 2 of 10 animals.

^d Estrous cycle was longer than 12 days or unclear in 6 of 10 animals.

^e Evidence shows that females exposed to 125 mg/kg differ significantly (Wilkes’ Criterion, P≤0.05) from the vehicle control females in the relative length of time spent in the estrous stages. Tests for equality of transition probability matrices among all groups and between the vehicle control group and each dosed group indicated that females in the 250 mg/kg dose group spent significantly (P<0.001) more time in extended diestrus than females in the vehicle control group.

Applicant's summary and conclusion

Conclusions:

No significant effects on spermatid or epididymal spermatozoal measurements were observed at any examined dose level (i.e. at 0, 62.5, 125 or 250 mg/kg). Decreased epididymis and testis weights were observed in male rats treated at 250 mg/kg; however no histopathological effects attributed to the test chemical were reported in testis (with epididymis and seminal vesicle). Oestrous cycle lengths were significantly increased at 125 mg/kg (mean, 5.56 days) and 250 mg/kg (mean 5.38 days) compared to the control group (mean 4.95). The numbers of cycling female rats at 0, 62.5, 125 or 250 mg/kg were 10, 10, 9 and 4, respectively. The numbers of females with regular oestrous cycle at 0, 62.5, 125 or 250 mg/kg were 9/10, 10/10, 8/9, and 4/4, respectively.The study-derived LOAEL for systemic toxicity (both genders) was 62.5 mg/kg bw. The observed effects on dioestrus in female rats treated at 125 and 250 mg/kg were not considered to be secondary to systemic toxicity.

Executive summary:

The chemical was given by oral gavage to 10 rats per sex per dose level at 0 (vehicle), 62.5, 125, 250, 500 and 1000 mg/kg bw (5 times per week) for a total of 14 weeks (core study rats). Additional groups (10 rats/sex/dose) treated as described above, but for 25 days, were also included in the study protocol (clinical pathology study). The animals were observed twice day and weighed once per week. Blood samples were collected on day 25 (clinical pathology rats) and on day 88 (core study rats). Haematological parameters included: haematocrit, haemoglobin, methaemoglobin concentrations, erythrocytes, reticulocytes, nucleated erythrocytes, platelets, Heinz body counts, MCV, mean cell haemoglobin, mean cell haemoglobin concentrations, leukocyte count, and leukocyte differentials. Clinical chemistry parameters included urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatinine kinase, sorbitol dehydrogenase, and bile acids. Spermatid and sperm samples were collected from male rats treated at 0, 62.5, 125 and 250 mg/kg, with the following parameters investigated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected at 0, 62.5, 125 and 250 mg/kg for up to 12 consecutive days prior to the end of treatment. All core study animals were necropsied, and the following organs were weighed: heart, right kidney, live, lung, right testis, and thymus. In addition to lesions, the following organs were examined histologically to a no-effect-level: adrenal gland, bone (including marrow), brain, clitoral gland, oesophagus, heart (including aorta), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung (and mainstem bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen,stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. All rats treated at 1000 mg/kg died by study day 3. With the exception of one male rat treated at 500 mg/kg, all rats treated at ≤500 mg/kg survived to planned death. Clinical signs of toxicity were observed at ≥250 mg/kg and included cyanosis, abnormal breathing, and lethargy. Significant decreases in terminal body weight were observed at ≥62.5 mg/kg. Male rats treated at 62.5, 125, and 250 mg/kg weighed approx. 9.5, 11.6, and 22.9% less, respectively, compared to the control group by the end of the study period. The corresponding decreases for female rats treated at 62.5, 125, and 250 mg/kg were 5.2, 10.8, and 9.8%, respectively. Methemoglobinemia and effects secondary to methemoglobinemia were observed at ≥62.5 mg/kg in both genders. No significant effects on spermatid or epididymal spermatozoal measurements were observed at any examined dose level (i.e. at 0, 62.5, 125 or 250 mg/kg). Decreased epididymis and testis weights were observed in male rats treated at 250 mg/kg; however no histopathological effects attributed to the test chemical were reported in testis (with epididymis and seminal vesicle). Oestrous cycle lengths were significantly increased at 125 mg/kg (mean, 5.56 days) and 250 mg/kg (mean 5.38 days) compared to the control group (mean 4.95). The numbers of cycling female rats at 0, 62.5, 125 or 250 mg/kg were 10, 10, 9 and 4, respectively. The numbers of females with regular oestrous cycle at 0, 62.5, 125 or 250 mg/kg were 9/10, 10/10, 8/9, and 4/4, respectively. At necropsy, increased liver weights (both genders) and increased kidney weights (females) were observed at ≥62.5 mg/kg. Histological findings included pigmentation of the liver at ≥62.5 mg/kg; hepatocyte hypertrophy at ≥125 mg/kg; hepatocyte necrosis at ≥62.5 mg/kg; degeneration of olfactory epithelium at ≥62.5 mg/kg; hyperplasia and metaplasia of the respiratory epithelium of the nose at ≥125 mg/kg; capsule fibrosis, congestion, hypertrophy, lymphoid follicle atrophy of the spleen at ≥125 mg/kg; nephropathy, papillary necrosis and mineralisation of the kidneys at ≥125 mg/kg. The study-derived LOAEL for systemic toxicity (both genders) was 62.5 mg/kg bw. The observed effects on dioestrus in female rats treated at 125 and 250 mg/kg were not considered to be secondary to systemic toxicity by the authors of the study. The study was performed according to GLP and was considered to be reliable with restrictions (Klimisch 2).