N,N-dimethyl-p-toluidine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Study 1 (sub-chronic toxicity study with the substance):

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. The dose of 62.5 mg/kg bw (which was given five days per week in the study) corresponds to a dose of about 44.6 mg/kg bw/day.

Study 2: OECD 422 study with read-across substance

NOAEL for systemic toxicity in male rats was considered to be 30 mg/kg bw/day based on adverse effects in clinical signs, haematology, clinical chemistry, gross pathology, and histopathology at 100 mg/kg bw/day. NOAEL for systemic toxicity in female rats could not be established due to lacking nursing activity and histopathologic lesions at 30 mg/kg bw/day. NOAEL for reproductive performance was considered at 300 mg/kg bw/day in both genders. NOAEL for developmental toxicity was considered at 30 mg/kg bw/day based on observed trend effects on live birth index and viability at 100 mg/kg bw/day. The observed trend effects on development were observed in the presence of maternal systemic toxicity that was characterised by lacking nursing activity, clinical signs of toxicity, gross findings in liver, spleen, kidneys, and histopathological lesions in the kidneys.

Link to relevant study records

Reference

Reference 1

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

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

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

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).

Dose / conc.:

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

Remarks:

Vehicle alone (corn oil)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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: 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

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

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)

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)

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)

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

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

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.

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).

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Females: Nulliparous and non-pregnant
- Age at study initiation: 7 weeks
- Weight at study initiation: Females 200.1 to 247.2 g; Males 267.2 to 361.7g
- Housing: Each animal was kept in a metal wire mesh floor cage (Japan cage) individually in the breeding room. A metal floorboard was laid on the floor of the breeding cage, and a wooden chip (white flake (R) and Nippon Charles River) were supplied as an appropriate floor covering for the mother animal after 18 days of pregnancy.
- Diet (e.g. ad libitum): solid feed (CA-1, Nippon Clare), ad libitum
- Water (e.g. ad libitum):tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%): relative humidity of 55±5%
- Air changes (per hr): ventilation time of about 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours of illumination (7:00 a.m. to 7:00 p.m.)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The prepared samples were sealed and stored under cool and dark conditions and administered within 7 days after preparation. Daily administration was carried out at a certain time (usually from 13:00 to 16:00) during a certain time, and the amount of administration of each animal was calculated based on the measured weight once a week for males and females before and during mating, and based on the weight of 0 days of pregnancy for females after mating. Dosing volume was set to 5 ml/kg bw.

Details on mating procedure:

Mating was carried by housing one male with one female (M/F ratio of 1:1). Evidence of copulation was checked every morning by checking the presence of sperm in the vaginal plug. When sperm was detected in the vaginal plug, it was considered as gestation day 0. Each female in gestation day 0 was separated from the male rat and raised individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

It was confirmed that the test chemical in the preparation solution was stable for at least 7 days under cool and dark conditions, and that the samples used contained almost a predetermined amount of the test chemical

Duration of treatment / exposure:

Male rats were dosed 14 days before mating and thereafter, for a total of 42 days. Female rats were dosed 14 days before mating and thereafter to day 4 of lactation.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle: corn oil

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

13 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of a 2-week dose range finding study. Animals in the main study were allocated to the different treatment groups by stratified randomization (by considering body weight) so that there would be no significant difference in body weight between the groups prior to dose administration. Daily administration was carried out at a certain time (usually from 13:00 to 16:00) during a certain time, and the amount of administration of each animal was calculated based on the measured weight once a week for males and females before and during mating, and based on the weight of 0 days of pregnancy for females after mating.

Positive control:

None.

Parental animals: Observations and examinations:

Clinical signs, body weight and food intake:
All animals were observed daily for clinical signs. Body weight and food intake were recorded initially, once per week, on gestation day 0, 7, 14, and 20, and on lactation day 0 and 4.

Hematology and clinical chemistry:
Blood samples for haematology and clinical chemistry were collected from males only. Hematologic parameters included the number of red blood cells (RBC), leukocyte count (WBC), blood pigment concentration (Hb), average red blood cell volume (MCV), hematocrit value (Ht), average red blood cell blood clot concentration (MCCH), platelet count multi-item blood automatic measuring machine (Coul Counter Model S-PLUS IV) was measured. In addition, leukocyte classification was performed by observing blood smear specimens (Wright-Giemsa staining) by optical microscope. In all cases, following the blood collection for hematologic testing, heparin was collected using anticoagulants, and plasma was separated using centrifugal bioechemical automatic analyzer (COBAS-FARA) and Na-K-Cl analyzer-IT-3, total protein concentration (biuret method), albumin concentration (BCG method), total cholesterol concentration (COD/DAOS method), respectively, using centrifugal bioechemical automatic analyzer (COBAS-FARA) and Na-K-Cl analyzer-IT-3 type, total protein concentration (biuret method), and total cholesterol concentration (COD/DAOS method), Glucose concentration (glucokinase-G6PDH method), urea nitrogen concentration (Urease-Gl.DH method), fluorifine concentration (Jaffe method), alkaline phosphatase activity (p-nitrophenylphosphate substrate method), GOT activity (SSCC method), GPT (SSCC method), total bilirubin concentration (bilirubine); calcium concentration (OPCP method), inorganic phosphorus concentration (molybdate direct method), gamma-GTP activity (gamma-glutamil-p nitroanilide substrate method), sodium concentration (ion electrode method), potassium concentration (ion electrode method), chlorine concentration (electrometallic titrating method), and A/G ratio (calculation).

Reproductive parameters:
Reproductive parameters included number of mated pairs, number of copulated pairs, number of pregnant animals, fertility index, and pairing days until copulations.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

Developmental parameters included gestation index, gestation length, number of corpora lutea, number of implantation sites, implantation index, number of pups born, delivery index, number of pups alive (day 0 and 4 of lactation), birth index, live birth index, pup weight (day 0 and 4 of lactation), live litter size (day 0 and 4 of lactation), sex ratio, pup viability.

Postmortem examinations (parental animals):

All animals were necropsied, and the weight of the following organs were recorded: brain, heart, spleen, adrenal gland, thymus, liver, kidney, testis and epididymis. Complete histopathological examinations were performed on all rats treated at 0 and 300 mg/kg. In case treatment-related were observed at 300 mg/kg, histopathological examinations were performed at lower doses to a no-effect-level.

Postmortem examinations (offspring):

The pups were examined for gross abnormalities, and the organs in the thoracic cavity and abdominal cavity were fixed in ethanol and examined

Statistics:

The rate of mating and conception were evaluated by the chi^2 test. All other data were tested for the uniformity of the variance of each group by the Bartlett method, using one sample of the value obtained for each individual or the average value for each litter. As a result, when the variance was uniform, a one-way anova was performed, and when significance was observed between the groups, the difference in the mean values between the control group and each test substance administration group was tested by the Dunnett method or the Scheff method. If the variance was not uniform, a rank test of Kruskal-Wallis was performed, and the difference between the control group and each test chemical administration group was tested for Dunnett type or Scheff type when significance was observed between the groups. P < 0.05 was considered statistically significant.

Reproductive indices:

Copulation index, fertility index, gestation index, implantation index, delivery index

Offspring viability indices:

Birth index, live birth index, sex ratio, viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed at 30 mg/kg bw/day. Clinical signs at ≥100 mg/kg included brownish urine, low motor activity, pale extremities (at 300 mg/kg) and increased salivation.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant effects on average male body weight were observed, however male rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of study. No significant effects on average female body weight were observed during the premating period, however female rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of treatment (mean, 8.5 g at 300 mg/kg vs. mean, 19.4 g at 0 mg/kg). Significant effects on gestational body weights were observed at 300 mg/kg compared to the control groups. The average gestational body weights at 300 mg/kg were 7.2, 8.0 and 22.9% lower on day 7, 14 and 20, respectively, compared to the control group. This effect was accompanied by a significant decrease in body weight gain between gestation day 14 and 20 at 300 mg/kg (mean, 7.4 g) compared to the control group (mean 75.7 g). No statistically secured effects on average body weight or body weight gain were observed at 30 or 100 mg/kg during lactation. No tabular data on average body weight or body weight gain were presented at 300 mg/kg since no litters were produced at that dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant effects on food intake were observed at 300 mg/kg (both genders) during the first week of treatment only.

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):

The haematologic examination (performed in male rats only) showed evidence of anaemia at ≥100 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The clinical chemistry examination (performed in male rats only) showed changes indicate of liver dysfunction at ≥100 mg/kg

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings in the kidneys included pigmentation (both genders, 300 mg/kg), eosinophilic droplets in the proximal tubular epithelial cells (males, 100 and 300 mg/kg), mild degeneration of renal interstitial (males, 300 mg/kg), regenerated tubular cells in the cortex and medulla (females, 30, 100 and 300 mg/kg), and calcification in the renal pelvis (females, 30, 100 and 300 mg/kg).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lacking nursing activity was observed at 30 mg/kg (in 2 of 11 females) and 100 mg/kg (in 2 of 8 females).

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No significant effects on mating performance or fertility were observed at any dose level. Gestation indexes were 100, 100, 80 and 0% at 0, 30, 100 and 300 mg/kg, respectively. The decrease in gestation index at 300 mg/kg was statistically secured.

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

other: Lacking nursing activity

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

haematology

clinical biochemistry

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal developmental toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: gestation index, gestation length, number of corpora lutea, implantation sites, implantation index.

Key result

Dose descriptor:

LOAEL

Remarks:

Maternal developmental toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Gestation index

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Live birth indexes were 91.4, 86.9 and 70.7, and 0% at 0, 30, 100 and 300 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the live birth index tended to decrease with dose.

Viability indexes were 98.6, 82.6, and 75.3% at 0, 30, and 100 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the viability index tended to decrease with dose.

The combination of tendency towards lower live birth index and lower viability index at 100 mg/kg was taken as an adverse effect of treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically secured differences in body weight were observed at 30 or 100 mg/kg, but pups tended to be of lower weight at 100 mg/kg compared to the control data.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross or visceral abnormalities were observed in any of the pups at 30 or 100 mg/kg.

Histopathological findings:

not examined

Other effects:

not specified

Description (incidence and severity):

No significant difference in sex ratio was observed.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Tabular data presented below.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

other: sex ratio

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

other: live birth index

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

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

Table 1.Combined repeat dose and reproductive/ developmental toxicity screening test for m-Toluidine in rats. Summary of reproductive performance

Compounds	Vehicle control	m-Toluidine	m-Toluidine	m-Toluidine
Dose groups Mg/kg	0	30	100	300
Number of mated pairs	13	13	13	13
Number of copulated pairs	13	13	12	13
Copulation index     A)	100.0	100.0	92.3	100.0
Number of pregnant animals	11	12	10	11
Fertility index          B)	84.6	92.3	83.3	84.6
Pairing days until copulation Mean ± S.D	2.6 ± 1.2	2.3 ± 1.4	2.2 ± 1.1	2.9 ± 1.5

 

A)   Copulation index = (Number of copulated pairs / Number of mated pairs) X 100 : %

B)   Fertility index = (Number of pregnant animals / Number of copulated pairs) X 100: %

Vehicle control: corn oil

             

Table 2.Combined repeat dose and reproductive/ developmental toxicity screening test for m-Toluidine in rats. Summary of reproductive performance

Compounds	Vehicle control	m-Toluidine	m-Toluidine	m-Toluidine
Dose groups Mg/kg	0	30	100	300
Number of pregnant females	11	11 a)	10	11
Number of pregnant females with pups alive	11	11	8	0
Gestation index     A)	100.0	100.0	80.0	0.0**
Gestation length in days	22.8 ± 0.4 (11)	22.6 ± 0.5 (11)	22.4 ± 0.5 (8)	b)
Number of corpora lutea	21.2 ± 5.6 (11)	19.2 ± 5.2 (11)	18.2 ± 5.7 (10)	19.4 ± 4.6 (11)
Number of implantation sites	14.7 ± 4.5 (11)	14.6 ± 4.2 (11)	14.3 ± 5.5 (10)	14.3 ± 3.4 (11)
Implantation index B)	72.7 ± 26.7 (11)	78.1 ± 22.2 (11)	76.6 ± 27.3 (10)	75.7 ± 20.9 (11)
Day 0 of lactation
Number of pups born	12.6 ± 3.6 (11)	13.2 ± 4.2 (11)	10.8 ± 7.2 (10)	0.0 ± 0.0 ** (11)
Delivery index          C)	87.6 ± 10.4 (11)	89.3 ± 8.0 (11)	68.1 ± 41.1 (10)	0.0 ± 0.0 ** (11)
Number of pups alive	11.4 ± 3.4 (11)	11.1 ± 3.6 (11)	9.0 ± 6.3  (8)
Birth index               D)	80.8 ± 19.0 (11)	77.1 ± 16.1 (11)	58.1 ± 35.1 (8)
Live birth index       E)	91.4 ± 15.1 (11)	86.9 ± 18.0 (11)	70.7 ± 36.2 (8)
Sex ratio                   F)	57.2 ± 14.7 (11)	53.1 ± 17.4 (11)	48.6 ± 14.3 (7)
Day 4 of lactation
Number of pups alive	11.2 ± 3.3 (11)	9.4 ± 4.9 (11)	8.3 ± 6.3 (7)
Viability index          G)	98.6 ± 3.1 (11)	82.6 ± 32.6 (11)	75.3 ± 38.7 (7)

 

Parenthesis indicates Number of litters evaluated

A)   Gestation index = (Number of pregnant females with pups alive / Number of pregnant females) X 100 : %

B)   Implantation index = (Number of implantation sites / Number of corpora lutea) X 100 : %

C)   Delivery index = (Number of pups born / Number of implantation sites) X 100 : %

D)   Birth index = (Number of pups alive on day 0 / Number of implantation sites) X 100 : %

E)   Live birth index = (Number of pups alive on day 0 / Number of pups born) X 100 : %

F)   Sex ratio = (Number of male pups alive on day 0 / Number of pups alive on day 0) X 100 : %

G)  Viability index = (Number of pups alive on day 4 / Number of pups alive on day 0) X 100 : %

 

a)   One female was excluded from data analysis because leakage of water from water supply system in the lactation period.

b)   All litters showed total implantation loss

Vehicle control: corn oil

** ; Significant difference from control, P< 0.01

 

Table 3.Combined repeat dose and reproductive/ developmental toxicity screening test for m-Toluidine in rats.Body weight of F1 pups up to day 4 of lactation; Mean ± S.D.

Compounds	Vehicle control	m-Toluidine	m-Toluidine	m-Toluidine
Dose groups Mg/kg	0	30	100	300
Day 0 (At birth)
Live litter size	11.4 ± 3.4 (11)	11.1 ± 3.6 (11)	10.3 ± 5.5 (7)	a)
Male	6.5 ± 2.6	6.1 ± 3.0	5.0 ± 2.9
Female	4.9 ± 2.1	5.0 ± 1.9	5.3 ± 2.8
Pup weight in gms
Male	7.1 ± 0.8	6.6 ± 1.2	6.0 ± 0.5
Female	6.4 ± 0.5	6.1 ± 1.0	5.5 ± 0.6
Day 4
Live litter size	11.2 ± 3.3 (11)	10.3 ± 4.0 (10)	9.7 ± 5.7 (6)
Male	6.3 ± 2.4	5.7 ± 3.2	5.0 ± 3.0
Female	4.9 ± 2.1	4.6 ± 2.1	4.7 ± 2.8
Pup weight in grams
Male	11.2 ± 2.6	10.4 ± 3.3	8.9 ± 1.5
Female	10.5 ± 2.2	10.0 ± 3.0	8.5 ± 1.6

 

Parenthesis indicates the number of litters evaluated

a)   All litters showed total implantation loss

Vehicle control: corn oil             

 

Table 4.Combination repeat dose and reproductive/development toxicity screening test for m-Toluidine in rats. Summary of morphological observation of pups

Compound	Vehicle control	m-Toluidine	m-Toluidine	m-Toluidine
Dose Group (mg/Kg)	0	30	100	300
No. of Pups Examined
External Observation	123	103	58	a)
Visceral Observation	123	103	58
No. of Pup Showing abnormalities
External abnormalities	0	0	0
Visceral abnormalities
Major abnormalities	0	0	0
Minor abnormalities	0	0	0

 

a): All litters showed total implantation loss

Vehicle control: Corn oil

Conclusions:

NOAEL for systemic toxicity in male rats was considered to be 30 mg/kg bw/day based on adverse effects in clinical signs, haematology, clinical chemistry, gross pathology, and histopathology at 100 mg/kg bw/day. NOAEL for systemic toxicity in female rats could not be established due to lacking nursing activity and histopathologic lesions at 30 mg/kg bw/day. NOAEL for reproductive performance was considered at 300 mg/kg bw/day in both genders. NOAEL for developmental toxicity was considered at 30 mg/kg bw/day based on observed trend effects on live birth index and viability at 100 mg/kg bw/day. The observed trend effects on development were observed in the presence of maternal systemic toxicity that was characterised by lacking nursing activity, clinical signs of toxicity, gross findings in liver, spleen, kidneys, and histopathological lesions in the kidneys.

Executive summary:

The chemical was given by oral gavage to 13 rats per sex per dose level at 0 (vehicle; corn oil), 30, 100 and 300 mg/kg bw/day. Male rats were dosed 14 days before mating and thereafter, for a total of 42 days. Female rats were dosed 14 days before mating and thereafter to day 4 of lactation. Dose levels were selected based on the results of a dose-range finding study. All animals were observed daily for clinical signs. Body weight and food intake were recorded initially, once per week, on gestation day 0, 7, 14, and 20, and on lactation day 0 and 4. Blood samples for haematology and clinical chemistry were collected from males only. All animals were necropsied, and the weight of the following organs were recorded: brain, heart, spleen, adrenal gland, thymus, liver, kidney, testis and epididymis. Complete histopathological examinations were performed on all rats treated at 0 and 300 mg/kg. In case treatment-related were observed at 300 mg/kg, histopathological examinations were performed at lower doses to a no-effect-level. Reproductive parameters included number of mated pairs, number of copulated pairs, number of pregnant animals, fertility index, and pairing days until copulations. Developmental parameters included gestation index, gestation length, number of corpora lutea, number of implantation sites, implantation index, number of pups born, delivery index, number of pups alive (day 0 and 4 of lactation), birth index, live birth index, pup weight (day 0 and 4 of lactation), live litter size (day 0 and 4 of lactation), sex ratio, pup viability. The pups were examined for gross abnormalities, and the organs in the thoracic cavity and abdominal cavity were fixed in ethanol and examined. RESULTS (PARENTAL GENERATION): All animals survived to planned death. No clinical signs were observed at 30 mg/kg bw/day. Clinical signs at ≥100 mg/kg included brownish urine, low motor activity, pale extremities (at 300 mg/kg) and increased salivation. No significant effects on average male body weight were observed, however male rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of study. No significant effects on average female body weight were observed during the premating period, however female rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of treatment (mean, 8.5 g at 300 mg/kg vs. mean, 19.4 g at 0 mg/kg). Significant effects on gestational body weights were observed at 300 mg/kg compared to the control groups. The average gestational body weights at 300 mg/kg were 7.2, 8.0 and 22.9% lower on day 7, 14 and 20, respectively, compared to the control group. This effect was accompanied by a significant decrease in body weight gain between gestation day 14 and 20 at 300 mg/kg (mean, 7.4 g) compared to the control group (mean 75.7 g). No statistically secured effects on average body weight or body weight gain were observed at 30 or 100 mg/kg during lactation. No tabular data on average body weight or body weight gain were presented at 300 mg/kg since no litters were produced at that dose level. Significant effects on food intake were observed at 300 mg/kg (both genders) during the first week of treatment only. The haematologic examination (performed in male rats) showed evidence of anaemia at ≥100 mg/kg. Changes in clinical chemistry were observed at ≥100 mg/kg and were indicate of liver dysfunction. Increased extramedullary haematopoiesis, and iron-positive pigmentation in the liver, spleen, or kidneys were observed in both genders at ≥100 mg/kg. Histopathological findings in the kidneys included pigmentation (both genders, 300 mg/kg), eosinophilic droplets in the proximal tubular epithelial cells (males, 100 and 300 mg/kg), mild degeneration of renal interstitial (males, 300 mg/kg), regenerated tubular cells in the cortex and medulla (females, 30, 100 and 300 mg/kg), and calcification in the renal pelvis (females, 30, 100 and 300 mg/kg). No significant effects on mating performance or fertility were observed at any dose level. Gestation indexes were 100, 100, 80 and 0% at 0, 30, 100 and 300 mg/kg, respectively. The decrease in gestation index at 300 mg/kg was statistically secured. Lacking nursing activity was observed at 30 mg/kg (in 2 of 11 females) and 100 mg/kg (in 2 of 8 females). RESULTS (PUPS): Live birth indexes were 91.4, 86.9 and 70.7, and 0% at 0, 30, 100 and 300 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the live birth index tended to decrease with dose. Viability indexes were 98.6, 82.6, and 75.3% at 0, 30, and 100 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the viability index tended to decrease with dose. The combination of tendency towards lower live birth index and lower viability index at 100 mg/kg was taken as an adverse effect of treatment. No statistically secured differences in body weight were observed at 30 or 100 mg/kg, but pups tended to be of lower weight at 100 mg/kg compared to the control data. No gross or visceral abnormalities were observed in any of the pups at 30 or 100 mg/kg. No significant changes in sex ratio were observed. CONCLUSION: NOAEL for systemic toxicity in male rats was considered to be 30 mg/kg bw/day based on adverse effects in clinical signs, haematology, clinical chemistry, gross pathology, and histopathology at 100 mg/kg bw/day. NOAEL for systemic toxicity in female rats could not be established due to lacking nursing activity and histopathologic lesions at 30 mg/kg bw/day. NOAEL for reproductive performance was considered at 300 mg/kg bw/day in both genders. NOAEL for developmental toxicity was considered at 30 mg/kg bw/day based on observed trend effects on live birth index and viability at 100 mg/kg bw/day. The observed trend effects on development were observed in the presence of maternal systemic toxicity that was characterised by lacking nursing activity, clinical signs of toxicity, gross findings in liver, spleen, kidneys, and histopathological lesions in the kidneys.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

44.6 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 2

Effects on developmental toxicity

Description of key information

Study 2: OECD 422 study with read-across substance

NOAEL for systemic toxicity in male rats was considered to be 30 mg/kg bw/day based on adverse effects in clinical signs, haematology, clinical chemistry, gross pathology, and histopathology at 100 mg/kg bw/day. NOAEL for systemic toxicity in female rats could not be established due to lacking nursing activity and histopathologic lesions at 30 mg/kg bw/day. NOAEL for reproductive performance was considered at 300 mg/kg bw/day in both genders. NOAEL for developmental toxicity was considered at 30 mg/kg bw/day based on observed trend effects on live birth index and viability at 100 mg/kg bw/day. The observed trend effects on development were observed in the presence of maternal systemic toxicity that was characterised by lacking nursing activity, clinical signs of toxicity, gross findings in liver, spleen, kidneys, and histopathological lesions in the kidneys.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2

Additional information

Study 1:

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).

Study 2:

The chemical was given by oral gavage to 13 rats per sex per dose level at 0 (vehicle; corn oil), 30, 100 and 300 mg/kg bw/day. Male rats were dosed 14 days before mating and thereafter, for a total of 42 days. Female rats were dosed 14 days before mating and thereafter to day 4 of lactation. Dose levels were selected based on the results of a dose-range finding study. All animals were observed daily for clinical signs. Body weight and food intake were recorded initially, once per week, on gestation day 0, 7, 14, and 20, and on lactation day 0 and 4. Blood samples for haematology and clinical chemistry were collected from males only. All animals were necropsied, and the weight of the following organs were recorded: brain, heart, spleen, adrenal gland, thymus, liver, kidney, testis and epididymis. Complete histopathological examinations were performed on all rats treated at 0 and 300 mg/kg. In case treatment-related were observed at 300 mg/kg, histopathological examinations were performed at lower doses to a no-effect-level. Reproductive parameters included number of mated pairs, number of copulated pairs, number of pregnant animals, fertility index, and pairing days until copulations. Developmental parameters included gestation index, gestation length, number of corpora lutea, number of implantation sites, implantation index, number of pups born, delivery index, number of pups alive (day 0 and 4 of lactation), birth index, live birth index, pup weight (day 0 and 4 of lactation), live litter size (day 0 and 4 of lactation), sex ratio, pup viability. The pups were examined for gross abnormalities, and the organs in the thoracic cavity and abdominal cavity were fixed in ethanol and examined.RESULTS (PARENTAL GENERATION):All animals survived to planned death. No clinical signs were observed at 30 mg/kg bw/day. Clinical signs at ≥100 mg/kg included brownish urine, low motor activity, pale extremities (at 300 mg/kg) and increased salivation. No significant effects on average male body weight were observed, however male rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of study. No significant effects on average female body weight were observed during the premating period, however female rats treated at 300 mg/kg showed a significant decrease in body weight gain during the first week of treatment (mean, 8.5 g at 300 mg/kg vs. mean, 19.4 g at 0 mg/kg). Significant effects on gestational body weights were observed at 300 mg/kg compared to the control groups. The average gestational body weights at 300 mg/kg were 7.2, 8.0 and 22.9% lower on day 7, 14 and 20, respectively, compared to the control group. This effect was accompanied by a significant decrease in body weight gain between gestation day 14 and 20 at 300 mg/kg (mean, 7.4 g) compared to the control group (mean 75.7 g). No statistically secured effects on average body weight or body weight gain were observed at 30 or 100 mg/kg during lactation. No tabular data on average body weight or body weight gain were presented at 300 mg/kg since no litters were produced at that dose level. Significant effects on food intake were observed at 300 mg/kg (both genders) during the first week of treatment only. The haematologic examination (performed in male rats) showed evidence of anaemia at ≥100 mg/kg. Changes in clinical chemistry were observed at ≥100 mg/kg and were indicate of liver dysfunction. Increased extramedullary haematopoiesis, and iron-positive pigmentation in the liver, spleen, or kidneys were observed in both genders at ≥100 mg/kg. Histopathological findings in the kidneys included pigmentation (both genders, 300 mg/kg), eosinophilic droplets in the proximal tubular epithelial cells (males, 100 and 300 mg/kg), mild degeneration of renal interstitial (males, 300 mg/kg), regenerated tubular cells in the cortex and medulla (females, 30, 100 and 300 mg/kg), and calcification in the renal pelvis (females, 30, 100 and 300 mg/kg). No significant effects on mating performance or fertility were observed at any dose level. Gestation indexes were 100, 100, 80 and 0% at 0, 30, 100 and 300 mg/kg, respectively. The decrease in gestation index at 300 mg/kg was statistically secured. Lacking nursing activity was observed at 30 mg/kg (in 2 of 11 females) and 100 mg/kg (in 2 of 8 females).RESULTS (PUPS):Live birth indexes were 91.4, 86.9 and 70.7, and 0% at 0, 30, 100 and 300 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the live birth index tended to decrease with dose. Viability indexes were 98.6, 82.6, and 75.3% at 0, 30, and 100 mg/kg, respectively. No statistically secured effect was observed at 30 or 100 mg/kg, however the viability index tended to decrease with dose. The combination of tendency towards lower live birth index and lower viability index at 100 mg/kg was taken as an adverse effect of treatment. No statistically secured differences in body weight were observed at 30 or 100 mg/kg, but pups tended to be of lower weight at 100 mg/kg compared to the control data. No gross or visceral abnormalities were observed in any of the pups at 30 or 100 mg/kg. No significant changes in sex ratio were observed.CONCLUSION:NOAEL for systemic toxicity in male rats was considered to be 30 mg/kg bw/day based on adverse effects in clinical signs, haematology, clinical chemistry, gross pathology, and histopathology at 100 mg/kg bw/day. NOAEL for systemic toxicity in female rats could not be established due to lacking nursing activity and histopathologic lesions at 30 mg/kg bw/day. NOAEL for reproductive performance was considered at 300 mg/kg bw/day in both genders. NOAEL for developmental toxicity was considered at 30 mg/kg bw/day based on observed trend effects on live birth index and viability at 100 mg/kg bw/day. The observed trend effects on development were observed in the presence of maternal systemic toxicity that was characterised by lacking nursing activity, clinical signs of toxicity, gross findings in liver, spleen, kidneys, and histopathological lesions in the kidneys.

Justification for classification or non-classification

Treatment with the substance or the read-across substance in rats is associated with adverse effects on estrous cyclicity (at 125 and 250 mg/kg bw and above in the 14 -week study), gestation index (at 300 mg/kg bw in the OECD 422 study), live birth index (at 100 and 300 mg/kg bw in the OECD 422 study), and pup viability (at 100 and 300 mg/kg bw in the OECD 422). These effects were observed at dose levels that were clearly toxic to the adult or maternal rats, as evident by adverse effects on body weight, hematology (methemoglobinemia, anemia), histology of tissue (notably hepatocyte necrosis, degeneration of olfactory epithelium, capsule fibrosis, congestion, atrophy of the spleen) both at 62.5 and 125 mg/kg bw in the 14 -week study; and adverse effects on clinical signs (low motor activity), hematology (anemia), clinical chemistry (indicative of liver dysfunction), and histology (kidney lesions) at 100 mg/kg bw in the OECD 422 study. The observed effects on reproductive/developmental endpoints in both studies were only observed in the presence of maternal toxicity, however it is not likely that they were merely secondary or non-specific to poor maternal health status. The substance is therefore considered to be classified for reproductive toxicity using the following hazard classification: ”H361: suspected of damaging fertility or the unborn child". The read-across justification is attached in section 13.2 of this registration dossier.