1,3-di-o-tolylguanidine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Reproductive and developmental toxicity screening test of basic rubber accelerator, 1,3-di-o-tolylguanidine, in rats.

Author:

Ema M, Kimura E, Matsumoto M, Hirose A and, Kamata E

Year:

2006

Bibliographic source:

Reprod Toxicol. 2006, Jul; 22(1):30-6.

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 3

Reference Type:

publication

Title:

Reproductive/developmental toxicity screening test of 1,3-di-o-tolylguanidine in rats.

Author:

Ema M, Kimura E, Hirose A, and Kamata E.

Year:

2005

Bibliographic source:

Abstracts/ Toxicology Letters 158S, S1-S258

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-di-o-tolylguanidine

EC Number:

202-577-6

EC Name:

1,3-di-o-tolylguanidine

Cas Number:

97-39-2

Molecular formula:

C15H17N3

IUPAC Name:

1,3-di-o-tolylguanidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river japan co. Atsugi Breeding center
- Age at study initiation: 10 weeks of age
- Weight at study initiation: males 346.2-408.2g, females 216.9-260.5g
- Fasting period before study: no
- Housing: during the administration, animals were housed individually in stainless steel cages with screen floors.
- Diet (e.g. ad libitum): autoclaved sterilized solid food (CRF-1, Oriental yeast co. ltd), ad libitum
- Water (e.g. ad libitum): well water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):24+/-2°C
- Humidity (%):55+/-10%
- Air changes (per hr):13-15
- Photoperiod (hrs dark / hrs light):12/12 (lighed from 7:00am to 7:00pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: A solution of 0.1w/v% Tween 80 added to 0.5w/v% sodium carboxymethyl cellulose (CMC-Na)

Details on exposure:

With the administration volume of 10 mL/kg, the control group was administered the same volume of a solution of 0.5 w/v% CMC-Na to which 0.1 w/v% Tween 80 was added. The administration dose was based on the most recent body weights of the males and females before and during the mating period, and after copulation, calculated based on the body weights on day 0 of gestation.

Details on mating procedure:

Mating started around 4:00pm on day 15 of administration
- M/F ratio per cage: 1M/1F (12 weeks of age)
- Length of cohabitation: two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): in polycarbonate cages equipped with bedding

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males were subject to daily doses administered for 14 days of mating and then 35 days, for a total of 49 days.
Females were subject to daily doses administered for 14 days of mating, periods of mating (a maximum of 14 days until successful copulation), gestation and three days of lactation. The day that administration started was considered day 1 of administration.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The administration amount was set based on the results of the preliminary 14 day repeat administration toxicity study conducted using rats [administration amount: 10, 20, 40 and 80 mg/kg; number of animals: 5 of each sex per group (not conforming to GLP)]). In this study, all of the males and 4 females in the 80 mg/kg groups perished. Among those that perished, a reduction in spontaneous locomotor activity, bradypnea, mydriasis, lying prone, lying laterally and tremors were confirmed during observation of the general conditions. Among the survivors, one female in the 80 mg/kg group exhibited a reduction in spontaneous locomotor activity, bradypnea, mydriasis, tremors and salivation while two females in the 40 mg/kg group exhibited a reduction in spontaneous locomotor activity, mydriasis, tremors and salivation. Both sexes in the 80 mg/kg group and females in the 40 mg/kg group exhibited a significant reduction in the values for food consumption on day 2 of administration when compared to the control group. The necropsy findings showed dark red lungs in 4/5 males and 3/4 females from the 80 mg/kg group that perished. As a result, in this study, a maximum dose of 50 mg/kg was set where it was believed that clear toxicity could be detected, and subsequent doses were set to 20 and 8 mg/kg, using a ratio of 2.5. Additionally, a control group was established, using only the vehicle.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, twice daily
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:
BODY WEIGHT: Yes. Twice a week during the administration period for the males. For the females : twice a week during the administration period before mating and during the mating period and then on days 0, 7, 14 and 21 of gestation, and days 0 and day 4 of lactation.
FOOD CONSUMPTION : yes. Males = twice a week . Females = twice a week during the administration period before mating, and then on days 1, 7, 14 and 21 of gestation, and days 1 and 4 of lactation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no

Oestrous cyclicity (parental animals):

Examinations of the estrous cycles of females were performed by vaginal sampling for a given time each morning for 15 days from the first day of administration (day 1 of administration). During the examination of estrous cycles, the number of estrous cycles was calculated and the number of days in an estrous cycle was the number of days between estrous to the following estrous cycle, used to calculate the mean estrous cycle.

Sperm parameters (parental animals):

not examined

Litter observations:

Verification of delivery was limited to during the morning, and those delivering after noon were considered to have delivered on the following day. For those delivered, examinations were conducted on the number of pups delivered during delivery, the number of live pups, the number of stillborn pups, gender of live pups and external abnormalities. For the live pups, measurements were conducted on the body weights on the delivery day as well as on day 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE: no data
GROSS NECROPSY: yes, Gross necropsy consisted of external and internal examinations.
HISTOPATHOLOGY / ORGAN WEIGHTS: yes,
The weight of testes and epididymides of the males, and the ovaries of the females were measured.
Histopathology on these same organs.

Postmortem examinations (offspring):

All of the live pups on day 4 of lactation were subject to exsanguination under ether anesthesia, and visual observations made on the organs and tissues. Additionally, after necropsy, the pups delivered (including stillborn pups, fatalities and those with external abnormalities) were individually fixed for storage using pure ethanol.

Statistics:

The mean values and standard deviations were calculated for each group for body weight, food consumption, number of days required for copulation, examinations on the estrous cycles (estrous cycle and number of estrous cycles), organ weights, ratio of organ weights to body weight, gestation term, number of corpus luteum, number of implantation scars, total number of births, number of live births, and body weight of live births. Next, equal distribution examination was conducted between the control group and the test substance administration group using the Bartlett method. If there was equal distribution, comparisons were made with the control group using the Dunnett method for multiple comparison. On the other hand, if equal distribution was not confirmed, comparisons were conducted according to the Steel method of multiple comparison. In all of the cases, the level of significance was set to 1 and 5%, and tests were conducted using both. Additionally, comparisons were made between the control group and each administration group for the copulation rate, fertilization rate, delivery rate and gender ratio of live pups were compared using ¿2 calibration. The implantation rate, stillbirth rate, rate of external abnormalities, rate of appearance of external abnormalities in pups by type, birth rate and survival rate of live births on day 4 were compared using the Wilcoxon signed rank test. Histopathological examinations were compared using the Mann-Whitney u-test. In all of the cases, the level of significance was set to 1 and 5%. Measurement values for the live pups were in single quantities.

Reproductive indices:

Implantation rate, copulation rate, fertilization rate, gestation period, fertility rate

Offspring viability indices:

Survival rate, stillborn rate, survival rate of pups on day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Among the surviving males, mydriasis was noted in nearly all of the animals from day 1 to day 9 of administration, and in addition to these, bradypnea, a reduction in spontaneous locomotor activity and lying prone were noted in nearly all of the animals. The occurrence frequency of these symptoms decreased as the number of days of administration passed. Additionally, the tremors were seen in only a few animals and were sporadic, and salivation was noted sporadically in all animals from day 22 of administration until the final day of administration. In the 20 mg/kg group, salivation was noted sporadically in half of the animals from day 28 of administration until the final day of administration.
Among the surviving females, mydriasis, bradypnea, a reduction in spontaneous locomotor activity and lying prone were sporadically noted in nearly all animals from day 1 of administration until day 0 of lactation but the number of animals exhibiting these decreased as the number of days of administration passed. In the 20 mg/kg group, mydriasis, bradypnea, a reduction in spontaneous locomotor activity and lying prone were noted in one animal on both day 2 and day 3 of administration. In addition to these symptoms, half of the animals in the 50 mg/kg group experienced sporadic tremors from day 1 of administration until day 5 of gestation. Furthermore, sporadic salivation was noted in nearly all of the animals in the 50 mg/kg group from day 4 of gestation until day 3 of lactation and nearly half of the animals in the 20 mg/kg group from day 14 of gestation until day 3 of lactation.
These symptoms were evident in both sexes from the time immediately after administration for a period of four hours, but had disappeared by the following day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one fatality on day 6 and day 7 of administration among males in the 50 mg/kg group, two fatalities on day 1 of administration and one fatality on the first day of delivery among females. These fatalities exhibited mydriasis, a reduction in spontaneous locomotor activity, bradypnea, lying prone and tremors from approximately 10 to 20 minutes after administration, which indicates clonic convulsions and breathing difficulties, and death occurred at the earliest, approximately 20 minutes after administration, and about 3 ½ hours after administration at the latest. Furthermore, in the one female that perished on the delivery day, salivation was noted immediately after administration, in addition to the other symptoms.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly low values compared to the control group were noted in males in the 50 mg/kg group from day 4 to day 50 of administration.
A trend of low values compared to the control group was noted in females in the 50 mg/kg group from day 4 to day 50 of administration and significantly low values compared to the control group were noted from day 8 of administration throughout the gestation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly low values compared to the control group were noted in males in the 50 mg/kg group from day 2 to day 15 of administration.
A trend of low values compared to the control group was noted in females in the 50 mg/kg group from day 2 to day 11 of administration and significantly low values compared to the control group were noted from day 7 of gestation until day 4 of lactation. Also, significantly low values compared to the control group were noted in the 20 mg/kg group from day 4 to day 8 of administration. Among males in the 20 mg/kg group on day 39 of administration and females on day 21 of gestation, as well as days 1 and 4 of lactation, high values were noted but no changes in body weights were noted and since the changes noted were identical to those in both sexes in the 50 mg/kg group, these changes were believed to be incidental and unrelated to administration of the test substance.

Food efficiency:

not specified

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Among live males, slight atrophy of the seminiferous tubule in the testes was noted in one animal in both the control and 50 mg/kg groups. Among live females, an ovarian cyst was noted in one animal in the control group. Otherwise, no abnormalities were noted in the animals that perished, the animals that copulated or the sterile animals of both sexes.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

During the examinations of estrous cycles, no significant differences between the test substance administration groups and the control group were noted.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

During the examinations of reproductive performance, copulation was confirmed in all but one pair in the 8 mg/kg group, and pregnancy was confirmed in all but one pair in the 20 mg/kg group. The copulation rates for the control, 8, 20 and 50 mg/kg groups were 100%, 91.67%, 100% and 100% respectively. The fertilization rates were 100%, 100%, 91.67% and 100% respectively, and thus, no significant differences between the test substance administration groups and the control group were noted. Additionally, no significant differences between the test substance administration group and the control group were noted for the number of days required for copulation. Among the animals in the 8 mg/kg group that did not copulate and the sterile animals in the 20 mg/kg group, no changes were noted during the necropsy and histopathological examination of the reproductive organs of both sexes, and there were no findings that suggest deficiencies in the fertilization function.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(maternal toxicity)

Effect level:

8 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Mortalities are observed at 50 mg/kg bw/d, and severe clinical signs at 20 mg/kg bw/d.

2

Dose descriptor:

NOAEL

Remarks:

(reproductive performance)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were noted during the necropsy on all of the stillborn and live pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

Among live females, an ovarian cyst was noted in one animal in the control group as well as one of the stillborn pups in the 50 mg/kg group.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

During the examinations at delivery, significantly low values were noted for the number of pups delivered, number of newborns, birth rate and body weight of male and female newborns at day 0 after delivery in the 50 mg/kg group compared to that in the control group.
During the examination on the appearance of the newborns in the 50 mg/kg group, three animals had missing toes, six animals had bent tails and one animal had a short tail, missing toes and anal atresia, and so there were significantly higher values on the appearance rate of external abnormalities when compared to the control group.
There were no significant differences between the control group and the test substance administration groups for the gestation term, number of corpus luteum, number of implantation scars, implantation rate, stillborn rate, birth rate, gender ratio of live pups, and rate of appearance of external abnormalities by type.
During the examination of the lactation period, significantly lower values were noted in the 50 mg/kg group for survival rate of pups at day 4 after birth when compared to the control group.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

20 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: At 50 mg/kg bw/d, external abnormalities are observed in pups.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Absolute and relative organ weights of rats treated orally with DOTG in the preliminary reproduction toxicity screening test

 

Dose (mg/kg)		0	8	20	50
Male
No animals		12	12	12	10
Absolute organ weights	Final bw (g)	536.7+/-33.5	543.9+/-33.3	531.5+/-30.7	485.4+/-34.6**
	Epididymides (g)	1.16+/-0.10	1.21+/-0.19	1.21+/-0.12	1.23+/-0.07
	Testes (g)	3.24+/-0.34	3.34+/-0.19	3.31+/-0.28	3.30+/-0.24
Relative organ weights	Epididymides (g/100 g bw)	0.22+/-0.02	0.22+/-0.02	0.23+/-0.03	0.25+/-0.02**
	Testes (g/100 g bw)	0.60+/-0.05	0.62+/-0.07	0.63+/-0.07	0.68+/-0.07*
Female
No animals		12	11	11	6
Absolute organ weights	Final bw (g)	340.4+/-18.0	345.8+/-18.7	354.5+/-19.2	318.9+/-24.3
	Ovaries (g)	101.3+/-7.5	105.9+/-6.3	100.7+/-10.6	102.4+/-10.2
Relative organ weights	Ovaries (g/100 g bw)	29.8+/-2.0	30.6+/-1.5	28.4+/-2.8	32.1+/-2.4

* p< 0.05, **p< 0.01 (significantly different from control)

Values are mean +/-SD.

 

Table 2: Histopathological findings reproductive organs of rats treated orally with DOTG in the preliminary reproduction toxicity screening test

dose	0 mg/kg				8 mg/kg				20 mg/kg				50 mg/kg
Organs and findings	-	+	++	+++	-	+	++	+++	-	+	++	+++	-	+	++	+++
Males
Testis	(12)				(1)				(1)				(10)
àAtrophy,seminiferous tubule	11	1	0	0	1	0	0	0	1	0	0	0	9	1	0	0
Epididymis	NR (12)				NR (1)				NR (1)				NR (10)
Female
Ovary	(12)				(0)				(0)				(6)
Cyst, luteal	11	1	0	0									6	0	0	0

 

Not significantly different from control.

Grade sign: - = none, + = slight, ++ = moderate, +++ = severe

NR = no remarkable changes

Figures in parentheses are number of animals with tissues examined histopathologically.

8 mg/kg: uncopulating male

20 mg/kg: non-pregnant male

 

 

Table 3: Reproductive performance of rats treated orally with DOTG in the preliminary reproduction toxicity screening test

 

Doses	0 mg/kg	8 mg/kg	20 mg/kg	50 mg/kg
No. females examined	12	12	12	10
Count of estrus (a)	4.17+/-0.39	3.92+/-0.51	3.67+/-0.65	3.90+/-0.57
Estrous cycle (b)	4.01+/-0.18	4.06+/-0.31	4.08+/-0.29	4.13+/-0.22
No. of mated (male)	12	12	12	10
No. of mated (female)	12	12	12	10
No. of copulated (male) (c)	12 (100)	11 (91.67)	12 (100)	10 (100)
No. of copulated (female) (c)	12 (100)	11 (91.67)	12 (100)	10 (100)
No. of impregnated d)	12 (100)	11 (100)	11 (91.67)	10 (100)
No. of pregnant (c)	12 (100)	11 (100)	11 (91.67)	10 (100)
Duration of mating (b)	3.00+/-0.95	2.73+/-1.01	2.42+/-1.08	2.20+/-1.03

 

Not significantly different from control.

(a) Values are mean +/- SD

(b) Values are mean +/- SD (day)

(c) Values in parentheses represent percentages to the number of mated.

(d) Values in parentheses represent percentages to the number of copulated.

Applicant's summary and conclusion

Conclusions:

The NOAELs of DOTG for general and developmental toxicity in rats are 8 and 20 mg/kg bw/day, respectively.

Executive summary:

As a part of the toxicity studies relating to the existing OECD chemical substance safety inspections, DOTG was administered orally at 0 (solvent control), 8, 20 and 50 mg/kg/day to rats of both sexes (12 animals per group) for a period of 14 days prior to mating. Males were subject to administration for a period of 35 days, including the mating period, and females were subject to the mating period, the period of gestation and up to day 3 of lactation. We studied the repeated administration toxicity and reproductive performance on the parent animals as well as the impact on the development and growth of the pups.

 

Repeated Dose Toxicity

The deaths of two males and three females in the 50 mg/kg group were confirmed. Among the surviving animals, salivation was noted in both sexes in the 20 and 50 mg/kg group, along with mydriasis, a reduction in spontaneous locomotor activity, bradypnea, lying prone and tremors were noted in the females in the 20 mg/kg group and both sexes in the 50 mg/kg group. These symptoms were primarily seen on the day of administration, and the number of animals exhibiting these decreased with the passage of time after several days of administration, but salivation was noted sporadically throughout the administration period until the final day of administration. Furthermore, both sexes in the 50 mg/kg group experienced low values for body weight and food consumption, and females in the 20 mg/kg group exhibited low values for food consumption. The necropsy, organ weights and histopathological examination did not verify any impact of the administration of the test substance.

As indicated above, administration of the test substance in the 20 and 50 mg/kg groups did have an impact, so a no observable adverse effect level for repeat dose toxicity of 8 mg/kg/day is recommended for both sexes.

 

Reproduction/Developmental Toxicity

Administration of the test substance did not demonstrate an effect on the reproductive functions of parent animals, specifically, the estrous cycle, number of corpus luteum, number of implantations, implantation rate, copulation rate, fertilization rate, and number of days required for copulation. Examinations during delivery revealed low values for the number of pups delivered, number of live pups, birth rates and live pup body weight at day 0 after birth in both sexes as well as high values for the rate of appearance of external abnormalities. No impact by the administration of the test substance was noted for the gestation period, stillbirth rate, fertility rate, gender ratio of pups and the rate of appearance of external abnormalities by type. The examinations during lactation revealed low values for the survival rate of new pups on day 4 after birth.

As indicated above, while no impact on the reproductive performance of parent animals was exhibited by administration of the test substance, an impact was noted on the development and growth of the pups in the 50 mg/kg group, however at this dose a severe maternal toxicity was observed. Therefore, under the conditions of this study, the no observable adverse effect level of 50 mg/kg/day is recommended for reproductive performance in parent animals while the no observable adverse effect level of 20 mg/kg/day is recommended for developemental toxicity.