1,3-di-o-tolylguanidine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening reproduction test in available on DOTG (OECD 421, 2006). This study suggests that DOTG may be teratogenic at maternotoxic doses, but doesn't affect the fertility. The NOAEL of DOTG for reproduction performance is 50 mg/kg bw because no effect on fertility was observed in this study.

Link to relevant study records

Reference

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

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 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)

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

Dose / conc.:

8 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day

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

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.

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: 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.

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.

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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.

Critical effects observed:

no

Reproductive effects observed:

no

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The OECD 421 study is a reliable study performed according to the OECD 421 guideline and GLP compliance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Summary of the screening reproduction test (OECD 421, Ema 2006) :

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 gestation period and up to day 3 of lactation. The repeated administration toxicity and reproductive performance were studied on the parent animals as well as the impact on the development and growth of the pups.

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.

 

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.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 developmental toxicity.

Effects on developmental toxicity

Description of key information

A developmental toxicity study on rat in available on DOTG (OECD 414, 2006). This study suggests that DOTG is teratogenic at maternal toxic doses and the NOAELs of DOTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co.
- Age at study initiation: males = 11 weeks, females = 10 weeks
- Weight at study initiation:males = 354-401 g, females = 202-246 g
- Fasting period before study: no data
- Housing: 4 during the acclimatation period, 2 during the mating, then individually
- Diet (e.g. ad libitum): free access to sterilized solid food CRF-1 (oriental Yeast Co. Ltd) from metal food dishes
- Water (e.g. ad libitum): sapporo municipal water, ad libitum
- Acclimation period: 5 days prior the mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3°C
- Humidity (%):50+/-20%
- Air changes (per hr):10-15/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC-Na solution with 0.1% Tween 80 added

Details on exposure:

Administration volume = 5 ml/kg
The administration amount was set on the results of the 28 day repeat oral toxicity test and the simple reproductive toxicity study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test substance in the administration solutions for all of the concentrations during the first and last preparations were analyzed with HPLC, Millenium 32 chromatography manager, digital balance, compact refrigerated centrifuge, desktop ultrasonic cleaner, pH meter.

Details on mating procedure:

- Impregnation procedure: cohoused
The vagical mucous was sampled and observations were made on the estrous cycle under a microscope, and then one males and one female were placed together fot one night just before estrous.
- M/F ratio per cage: 1/1
- Length of cohabitation: one night. Mating occurred over a span of 6 days.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD6-GD19

Frequency of treatment:

Daily

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS + DETAILS CLINICAL OBSERVATIONS: Yes. The presence of dead animals and general observations on all animals were conducted twice daily (before and after administration) during the administration period, and at a frequency of once a day during the period before administration was started, and on the day of the necropsy.
BODY WEIGHT: Yes, on day 1, 3 and 6-20 of gestation
FOOD CONSUMPTION : Yes, on day 1, 3, 6, 9 12, 15, 18 and 20 of gestation
WATER CONSUMPTION : no
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Brain (cerebrum and cerebellum), pituitary gland, thymus, thyroid gland (left/right), epididymis (left/right), adrenals (left/right), spleen, heart, liver, lungs (including the trachea), kidneys (left/right), ovaries (left/right), uterus (fundus and cervix), vagina, mammary gland (right side) and places where visual abnormalities were noted (including the boundary areas with normal tissues).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of dead foetuses : Yes
- Number of live foetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data

Statistics:

See below

Indices:

Implantation rate, fetal survival rate, embryo/fetus death rate, sex ratio.
Rate of foetuses with external abnormalities, frequency of litters with foetuses with external abnormalities.
Rate of foetuses with internal abnormalities, frequency of litters with foetuses with internal abnormalities.
Progression of ossification, rate of skeletal variation in fetuses, rate of skeletal abnormalities in fetuses, frequency of litters with foetuses with skeletal variation, frequency of litters with foetuses with skeletal abnormalities

Historical control data:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During observation on the general conditions of maternal animals during the administration period, alopecia was noted on two animals each in the control group and the 10 mg/kg group. In the 20 and 40 mg/kg groups, 12 and 24 animals respectively experienced mydriasis between 10 and 50 minutes after administration, and a reduction in spontaneous locomotor activity was noted in 1 and 11 animals respectively. Also, the symptoms of lying prone (3 animals), bradypnea (2 animals) and tremors (2 animals) were noted in the 40 mg/kg group, and 4 animals (animal numbers 451, 463, 468 and 471) perished within 30 minutes after administration (days 8, 8, 19 and 7 of gestation). Among the surviving animals, these symptoms disappeared between 10 minutes and 2 hours after administration. As for other symptoms, there were 2 cases of salivation, 4 cases of soil in the perigenital fur and 2 cases of alopecia in the 40 mg/kg group. In the 20 mg/kg group, there were 2 cases of salivation, one case of soil in the perigenital fur and 3 cases of alopecia but there were no mortalities in the maternal animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the mean body weights and revised body weights of maternal animals in the 10 mg/kg group when compared to the control group. In the 20 mg/kg group, significantly low values were noted in the body weights on day 20 of gestation (necropsy day). In the 40 mg/kg group, significantly low values were noted in the body weights and the revised body weights from day 8 of gestation (day 3 of administration) until day 20 of gestation (necropsy day).
No significant differences were noted in the mean weight gain of maternal animals in the 10 mg/kg group when compared to the control group. In the 20 mg/kg group, all of the values were significantly low values on days 0-10 of gestation as well as from day 0-12 to day 0-20. In the 40 mg/kg group, significantly low values were noted throughout the administration period from day 0-7 of gestation to 0-20 of gestation. Significantly low values were noted in the 20 and 40 mg/kg groups for weight gain during the administration period (days 6-20 of gestation).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the mean food consumption amounts of maternal animals in the 10 and 20 mg/kg groups when compared to the control group. In the 40 mg/kg group, significantly low values were noted in the amount of food consumed during the administration period, on days 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation.

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):

effects observed, treatment-related

Description (incidence and severity):

see section "clinical signs"

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

During the necropsy performed on the maternal animals in the control group on day 20 of gestation, there was one case of situs inversus viscerum and two animals with alopecia, while there was one case of alopecia in each of the 10 and 20 mg/kg groups, and two cases of alopecia, two cases of thymus atrophy and one case of cystic kidney in the 40 mg/kg group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on maternal toxic effects:

Pregnancy was confirmed in all of the females in each group of 24 that successfully copulated, and with the exception of the females in the 40 mg/kg group that died, surviving fetuses were confirmed in all of these pregnant females. Significantly low values were noted for the gravid uterus weights in the 40 mg/kg group. Significantly high values were noted in the implantation rate in the 10 mg/kg group. Significantly different values were not noted between the control group and any of the test substance administration groups for the number of corpus luteum or number of implantations.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted for the number of dead fetuses, fetal death rate, number of surviving fetuses and fetal survival rate in the 10 and 20 mg/kg groups when compared to the control group. In the 40 mg/kg group, significantly high values were noted for the number of dead fetuses (both early stage and overall) and the fetal death rate while significantly low values were noted for the number of surviving fetuses and fetal survival rate.

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

Significantly low values were noted in all of the test substance administration groups for the fetal sex ratio.

Changes in litter size and weights:

effects observed, treatment-related

Changes in postnatal survival:

effects observed, treatment-related

External malformations:

effects observed, treatment-related

Description (incidence and severity):

During the external examination conducted on the surviving fetuses, there were 8 and 31 animals with short phalanges in the 20 and 40 mg/kg groups, respectively (the group means calculated from the rate of fetal external abnormalities for each litter were 2.17% and 14.38%), 7 and 10 animals with short tails (1.84% and 4.37%). In the 40 mg/kg group, the rate of incidence of short phalanges and short tails and the frequency in litters had significantly high values when compared to the values in the control group. Statistically significant differences were not noted in the 20 mg/kg group but a trend of high values was evident. Significantly high values were seen for the overall rate of fetuses with external abnormalities and the overall frequency in litters in the 40 mg/kg group. Additionally, one case of cleft palate was noted in the 10 mg/kg group (0.28%) but there were no significant differences in the rate of fetuses with abnormalities and the overall frequency in litters when compared to the values in the control group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal abnormalities in the 20 and 40 mg/kg groups manifested as missing tails, fusion or positional abnormalities were noted in 8 and 10 animals respectively (the group means calculated from the rate of fetal skeletal abnormalities for each litter were 4.17% and 8.56%). Missing or fused phalanges were noted in 5 and 18 animals respectively (2.60% and 16.02%). The rate of skeletal abnormalities in fetuses and the frequency in the litter exhibited significantly high values in the 40 mg/kg group when compared to those of the control group. Statistically significant differences were not noted in the 20 mg/kg group but a trend of high values was evident. In the 40 mg/kg group, fusion of the metacarpals/metatarsals and phalanges was noted in 2 animals (1.55%), missing or fused metacarpals was noted in 4 animals (3.38%), shortening of the tibia and fibula was noted in one animal (1.25%), and the rate of missing or fused metacarpals in fetuses and the frequency in the litter was significantly high. Thus, in the 20 and 40 mg/kg groups, significantly high values were noted for the overall rate of fetal skeletal abnormalities, and the 40 mg/kg group also had significantly high values for the overall frequency in the litter. Additionally, there was one animal (0.60% and 0.83%) in each of the 20 and 40 mg/kg groups with split cartilage of thoracic centrum, one animal (0.46%, 0.69% and 1.25%) in each of the 10, 20 and 40 mg/kg groups with fused cartilage of the cervical vertebral arches, and one animal (0.60%) in the control group with fused cartilage of the ribs. However, the rate of fetal skeletal abnormalities and the frequency in the litter for these did not exhibit significant differences between the test substance groups and the control group.
Skeletal variation was observed in all of the test groups as well as the control group. The type and the number of fetuses (the group means were calculated from the rate of skeletal variation for each litter) included 9 (4.94%), 12 (6.73%), 14 (8.06%) and 4 (2.89%) animals each in the control, 10, 20 and 40 mg/kg groups with short supernumerary ribs, 1 (0.60%) and 2 (1.43%) each in the control and 40 mg/kg group with unossified thoracic centrum, 2 (0.93%) and 1 (0.60%) each in the 10 and 20 mg/kg groups with bipartite ossification center of thoracic centrum, 1 (0.60% and 0.52%) each in the 10 and 20 mg/kg groups with wavy ribs, and 1 (0.60% and 1.25%) each in the 20 and 40 mg/kg groups with bipartitie ossification of sternebra. Additionally, there was one case (0.52%) of dumbbell ossification of the thoracic centrum in the 10 mg/kg group, 2 (1.43%) animals in the 40 mg/kg group with a short 13th rib, two (1.43%) animals in the 40 mg/kg group with sacralization of lumbar vertebra and one (0.83%) with asymmetry of sternebra. However, the rate of fetal skeletal variation and the frequency in the litter for these did not exhibit significant differences between the test substance groups and the control group.
Significant differences in the mean number for fetal vertebral ossification, thoracic centrum, and ossification of the metacarpals/metatarsals were not noted when the 10 and 20 mg/kg groups were compared with the control group. In the 40 mg/kg group, significantly low values were noted for ossification of the sacral and coccygeal vertebrae, thoracic centrum, and the number of ossified metacarpals and metatarsals.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For abnormalities with internal organs and tissues, there was one case in both the control group and the 20 mg/kg group with microphthalmia (0.60% and 0.52%), one case in the control group with dilatation of the lateral ventricle (0.60%), and one animal in the 40 mg/kg group with undescended testis (0.83%). However, no significant differences were noted in either the rate of abnormalities in fetal internal organs and tissues or the frequency in the litter when compared to the values of the control group.
Variation in the internal organs and tissues were noted in all of the test groups, including the control group. The number of fetuses (group mean calculated from the rate of variation in fetal internal organs and tissues for each litter) and their classifications included thymic remnant in the neck in 13 (7.52%), 8 (4.63%), 12 (6.87%) and 17 (14.80%) animals in the 10, 20 and 40 mg/kg groups, respectively, as well as left umbilical artery in 1 (0.69%), 1 (0.60%) and 2 (1.63%) animals, respectively, and dilatation of renal pelvis in 2 (1.22%) and 2 (1.35%) animals in the control group and the 10 mg/kg group, respectively. However, no significant differences were noted in either the rate of variation in fetal internal organs and tissues or the frequency in the litter when compared to the values of the control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the fetal weights and placenta weights for both sexes in the 10 and 20 mg/kg groups when compared to the control group, but significantly low values were noted for both in the 40 mg/kg group.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Remarks on result:

other: At 20 mg/kg bw/d: external malformations (short phalanges, short tails), skeletal malformations (missing tails, fusion or positional abnormalities).

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Maternal findings in rats given DOTG on days 6-19 of pregnancy

 

Dose (mg/kg)	0 (control)	10	20	40
No. of rats	24	24	24	24
No. of pregnant rats	24	24	24	24
Initial body weight	256+/-13	256+/-13	256+/-13	256+/-13
No. of females showing clinical signs of toxicity
Death	0	0	0	4
Alopecia	2	2	3	2
Bradypnea	0	0	0	2
Decreased locomotor activity	0	0	1	11**
Mydriasis	0	0	12	24**
Prone position	0	0	0	3
Salivation	0	0	2	2
Soil of perigenital	0	0	1	4
Tremor	0	0	0	2
Bodyweight gain during pregnancy (g) a)
Days 0-6	40+/-8	39+/-8	40+/-8	39+/-8
Days 6-15	50+/-7	49+/-9	37+/-11**	23+/-10**
Days 15-20	77+/-9	77+/-9	71+/-10	47+/-16**
Days 0-20	167+/-17	165+/-21	148+/-24**	109+/-21**
Adjusted weight gain b)	88+/-15	87+/-19	77+/-15	49+/-17**
Food consumption during pregnancy (g/day) a)
Days 0-6	23+/-2	23+/-2	23+/-2	23+/-2
Days 6-15	26+/-2	26+/-2	24+/-3	20+/-3**
Days 15-20	28+/-2	28+/-3	26+/-2	22+/-3**
Days 0-20	25+/-2	26+/-2	24/-2	21+/-2**
Weight of gravid uterus (g) a)	79+/-10	78+/-11	72+/-15	59+/-10**

a) values are given as the mean +/-SD.

b) adjusted weight gain refers to maternal weight gain excluding the gravid uterus.

** significantly different from the control (p<0.01).

 

 

Table 2: Reproductive findings in rats given DOTG on days 6-19 of pregnancy

 

Dose (mg/kg)	0 (control)	10	20	40
No. of litters	24	24	24	20
No. of litter totally resorbed	0	0	0	0
No. of corpora lutea per litter a)	15.7+/-2.1	14.8+/-1.6	14.9+/-1.9	15.3+/-1.5
No. of implantations per litter a)	15.3+/-1.9	14.7+/-1.8	14.2+/-2.7	15.2+/-1.4
% preimplantation loss per litter b)	2.4	0.9	5.6	0.9
% postimplantation loss per litter b)	3.5	3.4	4.8	16.4**
No. of live fetuses per litter a)	14.8+/-1.9	14.2+/-2.1	13.7+/-2.9	12.6+/-1.9**
Sex ratio of live fetuses (male/female)	0.56	0.49*	0.46*	0.46*
Bodyweight of live fetuses (g) a) :
-male	3.64+/-0.17	3.72+/-0.18	3.59+/-0.24	3.19+/-0.31**
-female	3.42+/-0.16	3.53+/-0.25	3.41+/-0.18	3.03+/-0.26**
Placental weight (g) a)	0.47+/-0.04	0.47+/-0.03	0.50+/-0.16	0.40+/-0.04**

Significantly different from the control (*: p< 0.05 and ** : p<0.01).

           

a)     values are given as the mean +/-SD.

b)      (No. of preimplantation embryonic loss/ no. of corpora lutea) x 100

c)     (No of resorptions and dead fetuses/ no. implantations) x 100

Conclusions:

Pregnant rats were given 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 10, 20 or 40 mg/kg bw/day on days 6-19 of pregnancy and the pregnancy outcome was determined on day 20 of pregnancy. At 40 mg/kg bw/day, deaths were observed in four out of 24 females. The incidences of females showing mydriasis at 20 and 40 mg/kg bw/day and showing decreased locomotor activity at 40 mg/kg bw/day were significantly increased. Alopecia, bradypnea, prone position and tremor were also observed at 40 mg/kg bw/day. The maternal body weight gain at 20 and 40 mg/kg bw/day and food consumption at 40 mg/kg bw/day were significantly reduced. A significantly decreased weight of the gravid uterus, increased incidence of postimplantation loss, decreased number of live fetuses, and lowered weights of fetuses and placentae were found at 40 mg/kg bw/day. The incidences of the total number of fetuses with external malformations at 40 mg/kg bw/day and with skeletal malformations at 20 and 40 mg/kg bw/day were significantly increased. Significantly higher incidences of fetuses with brachydactyly and short tail and defects of caudal vertebrae, phalanges and metacarpals were observed at 40 mg/kg bw/day. Delayed ossification was also noted at 40 mg/kg bw/day. The data indicate that DTG is teratogenic at maternal toxic doses and the NOAELs of DOTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.

Executive summary:

Using doses of 0 (control group), 10, 20 and 40 mg/kg/day of N, N’-bis (2-methylphenyl) guanidine administered orally using a gastric probe, the presence of toxicity on fetal rats was studied in 24 Crj (SD) IGS female rats per group with confirmed copulation from day 6 to day 19 of gestation.

During observations on the general conditions of the maternal animals, changes relating to administration of the test substance included mydriasis occurring 10 minutes to 50 minutes after administration in half of the animals in the 20 mg/kg group and all of the animals in the 40 mg/kg group. In the 40 mg/kg group, a reduction in spontaneous locomotor activity was also noted in approximately half of the animals. Additionally, four animals observed with the symptoms of lying prone, bradypnea and tremors died within 30 minutes after administration. The surviving animals recovered from these symptoms from about 10 minutes to two hours after administration. During the necropsy, changes believed to be related to administration of the test substance were not observed in any of the administration groups.

The body weights and amount of weight gain in the maternal animals did not exhibit significantly low values that correlated to the dose in either the 20 or the 40 mg/kg groups throughout the administration period. For food consumption, significantly low values were noted throughout the administration period in the 40 mg/kg group.

Examinations of the ovaries and uterus during the necropsy on day 20 of gestation revealed an impact on only the 40 mg/kg group: specifically, significantly low values for gravid uterine weight, significant increases in the number and rate of fetal mortalities, a significant reduction in the number and rate of surviving fetuses and significantly low values for fetal and placenta weights for both sexes. 

During the external examinations of the surviving fetuses, there was an increase in the rate of occurrence of short phalanges and short tails that correlated to the dose in the 20 and 40 mg/kg groups. Abnormalities of the skeletal frame included missing, damaged and fused tails, while positional abnormalities included missing, damaged and fused bones of the phalanges, with an increase in the rate of occurrence correlating to the doses in the 20 and 40 mg/kg groups. In the 40 mg/kg group, there was also fusion of the metacarpal/metatarsal and phalanx, and missing, damaged and fused metacarpal, and shortening of the tibia and fibula noted in 1~4 animals. On the other hand, there were no increases in abnormalities of the internal organs or tissues of the fetuses that related to administration of the test substance noted in any of the administration groups.

Relative to the fetal ossification, in the 40 mg/kg group, significantly low values were noted in the number of animals with ossification of the sacral and coccygeal vertebrae, thoracic centrum, and metacarpals and metatarsals.

 

From the abovementioned results and under the conditions of this study, the no observable adverse effect level of N, N’-bis (2-methylphenyl) guanidine on maternal and fetal rats is 10 mg/kg/day. On the other hand, clear toxicity was noted in maternal rats with doses of 20 and 40 mg/kg/day, which are believed to be doses that induce abnormalities of the four limbs and tails of fetuses. Additionally, the dose of 40 mg/kg/day is believed to be a dose at which there is an increase in the mortality rate of both maternal animals and fetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Ema's study is a reliable study performed according to the OECD 414 guideline and GLP compliance.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Summary of the developmental toxicity study on rat (OECD 414) :

Using doses of 0 (control group), 10, 20 and 40 mg/kg/day of DOTG administered orally using a gastric probe, the presence of toxicity on fetal rats was studied in 24 female rats per group with confirmed copulation from day 6 to day 19 of gestation.

During observations on the general conditions of the maternal animals, changes relating to administration of the test substance included mydriasis occurring 10 minutes to 50 minutes after administration in half of the animals in the 20 mg/kg group and all of the animals in the 40 mg/kg group. In the 40 mg/kg group, a reduction in spontaneous locomotor activity was also noted in approximately half of the animals. Additionally, four animals observed with the symptoms of lying prone, bradypnea and tremors died within 30 minutes after administration. The surviving animals recovered from these symptoms from about 10 minutes to two hours after administration. During the necropsy, changes believed to be related to administration of the test substance were not observed in any of the administration groups.

The body weights and amount of weight gain in the maternal animals did not exhibit significantly low values that correlated to the dose in either the 20 or the 40 mg/kg groups throughout the administration period. For food consumption, significantly low values were noted throughout the administration period in the 40 mg/kg group.

Examinations of the ovaries and uterus during the necropsy on day 20 of gestation revealed an impact on only the 40 mg/kg group: specifically, significantly low values for gravid uterine weight, significant increases in the number and rate of fetal mortalities, a significant reduction in the number and rate of surviving fetuses and significantly low values for fetal and placenta weights for both sexes. 

During the external examinations of the surviving fetuses, there was an increase in the rate of occurrence of short phalanges and short tails that correlated to the dose in the 20 and 40 mg/kg groups. Abnormalities of the skeletal frame included missing, damaged and fused tails, while positional abnormalities included missing, damaged and fused bones of the phalanges, with an increase in the rate of occurrence correlating to the doses in the 20 and 40 mg/kg groups. In the 40 mg/kg group, there was also fusion of the metacarpal/metatarsal and phalanx, and missing, damaged and fused metacarpal, and shortening of the tibia and fibula noted in 1~4 animals. On the other hand, there were no increases in abnormalities of the internal organs or tissues of the fetuses that related to administration of the test substance noted in any of the administration groups.

Relative to the fetal ossification, in the 40 mg/kg group, significantly low values were noted in the number of animals with ossification of the sacral and coccygeal vertebrae, thoracic centrum, and metacarpals and metatarsals.

 

From the abovementioned results and under the conditions of this study, the no observable adverse effect level of DOTG on maternal and fetal rats is 10 mg/kg/day. On the other hand, clear toxicity was noted in maternal rats with doses of 20 and 40 mg/kg/day, which are believed to be doses that induce abnormalities of the four limbs and tails of fetuses. Additionally, the dose of 40 mg/kg/day is believed to be a dose at which there is an increase i

Justification for classification or non-classification

Based on the available data, no classification for reproduction is required for 1,3-di-o-tolylguanidine according to the Regulation EC N°1272/2008.