1,3-di-o-tolylguanidine

Endpoint summary

Description of key information

Acute oral: Several studies are available for this endpoint, three studies are reliable and classified DOTG as toxic with a LD50 between 56 and 263 mg/kg bw. The key study is the study of 2003, with a LD50 of 56 mg/kg bw in rat.
Acute dermal: A GLP guideline study (OECD 402) of 2001 is available on rats. The dermal LD0 is higher to 2000 mg/kg bw.
Acute inhalation: no study is available.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

no

Test type:

fixed dose procedure

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: 6 weeks old
- Weight at study initiation: males = 177.4-190.2g, females = 131.4-152.7g
- Fasting period before study: yes 18 hours
- Housing: in polycarbonate cages with screen floors
- Diet (e.g. ad libitum): autoclaved sterilized solid food (CRF-1 Oriental yeast co), ad libitum
- Water (e.g. ad libitum):well water to which sodium hypochlorite had been added (2 ppm), ad libitum
- Acclimation period: 7 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-700pm)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Sodium carboxymethylcellulose solution mixed with 0.1 % Tween 80

Details on oral exposure:

The administration dose was 10 mL/kg, and the administration dose for each animal was calculated based on the weight on the administration day.
The same dose of the vehicle was administered to the control group.

Doses:

0 (Vehicle), 15, 30, 60, 120 mg/kg bw

No. of animals per sex per dose:

5 rats/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations : observations on the general conditions as well as the presence of mortalities were conducted continuously for six hours after administration on the administration day (day 0 of administration), twice a day in the morning and afternoon on days 1~13 of administration, and once a day during the morning on day 14 of administration.
- Frequency of weighing: Measurements were taken before administration on the day of administration (day 0 of administration), and then on days 1, 3, 5, 7, 10 and 14 after administration. Additionally, final weight measurements were made on animals that perished.
- Necropsy of survivors performed: yes
Using ether anesthesia, exsanguination was performed on the fatalities as soon as possible after verifying the deaths, and on all of the surviving animals on after the end of the observation period. One male from the 120 mg/kg group that perished (No. 25) exhibited visual abnormalities in the lungs, and one female in each the 60 and 120 mg/kg groups (No. 70 and 72) exhibited visual abnormalities in the stomach so these were extracted and fixed in 10% neutral buffered formalin. Additionally, paraffin embedded slices were created and hematoxylin and eosin (HE) staining conducted for observation under an optical microscope.

Statistics:

From the total number of fatalities after 14 days of administration, the LD50 value was calculated for the males using the Probit method and for the females using the Van der Waerden method. Mean values and standard deviations were determined for body weights for each group. No statistically significant differences were noted.

Preliminary study:

The administration amount was set based on the results of the preliminary single administration test conducted using rats [study number P020010 (not according to GLP), administration amount: 10, 30, 100 and 300 mg/kg; number of animals: 3 of each sex per group]. In this study, all of the males and females in the 100 and 300 mg/kg groups perished, and mydriasis, reduction in spontaneous activity, bradypnea and tremors were confirmed during observation of the general conditions. In this study, a maximum dose of 120 mg/kg was set where all of the cases were expected to perish, and subsequent doses were set to 60, 30 and 15 mg/kg, using a ratio of 2.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

85.3 mg/kg bw

Based on:

test mat.

95% CL:

> 44.8 - < 216.9

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

56 mg/kg bw

Based on:

test mat.

95% CL:

> 40.2 - < 78

Mortality:

There are no mortality at 15 and 30 mg/kg bw and in control group.
Deaths occurred in the males and females in the 60 mg/kg (1 male and 3 females) and 120 mg/kg groups (4 males and 5 females). At 60 mg/kg, the male and 2 females died 45 minutes after administration, and one female after 30 minutes. At 120 mg/kg, 4 males and all (5) females died after 30 minutes of administration.

Clinical signs:

other: Among the survivors, one male in the 120 mg/kg group exhibited the same symptoms 15 minutes after administration as those that perished, and was observed until 6 hours after administration, and all symptoms had disappeared by the day after administration

Gross pathology:

Visual examination of the males from the fatalities in the 120 mg/kg group revealed histological mild haemorrhaging in the lung where there was slight dark red coloration. Visual examination of the females from the fatalities in the 60 and 120 mg/kg groups revealed slight dark red spots on the proventriculus, and histologically, there was mild haemorrhaging noted in the stomach layer containing the proventriculus.

Other findings:

Necropsy : Of the mortalities, 1/4 males in the 120 mg/kg group exhibited slight dark red coloration of the lung. Additionally, 1/3 females in the 60 mg/kg group and 3/5 females in the 120 mg/kg group exhibited slight dark red spots on the proventriculus. There were no changes noted in any of the survivors.

Table 1: Mortality and lD50 values in rats after a single oral administration of DOTG

 

sex	Dose (mg/kg)	Number of animals	Time after administration						Total number of dead animals	LD50 (95% confidence interval) (mg/kg)
			Minutes				hours	Day
			0	15	30	45	1-6	1-14
male	0	5	0	0	0	0	0	0	0	85.3 (44.8-216.9)
	15	5	0	0	0	0	0	0	0
	30	5	0	0	0	0	0	0	0
	60	5	0	0	0	1	0	0	1
	120	5	0	0	4	0	0	0	4
female	0	5	0	0	0	0	0	0	0	56.0 (40.2 – 78.0)
	15	5	0	0	0	0	0	0	0
	30	5	0	0	0	0	0	0	0
	60	5	0	0	1	2	0	0	3
	120	5	0	0	5				5

 

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

The LD50 values are 85.3 mg/kg bw in males and 56 mg/kg bw in females.

Executive summary:

Single oral administration of doses of 0 (control), 15, 30, 60 and 120 mg/kg of N,N’-bis (2-methyl phenyl) guanidine was performed on five male and five female Crj:CD (SD) IGS rats in each group to study the acute toxic symptoms and the lethal dose. There are no mortality at 15 and 30 mg/kg bw and in control group.

Deaths occurred in the males and females in the 60 mg/kg (1 male and 3 females) and 120 mg/kg groups (4 males and 5 females). Treatment-related clinical signs were noted as follows: mydriasis, decrease in locomotor activity, bradypnea and tremors. Among males in the 60 mg/kg group, a trend of low values for body weights was noted on day 1 after administration and throughout the observation period in females in the 60 mg/kg group.

The LD50 values are 85.3 mg/kg bw in males and 56 mg/kg bw in females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

56 mg/kg bw

Quality of whole database:

The Honda's study is reliable with a klimisch score of 1 and was performed according to the guideline and GLP compliance.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-03-27 to 2001-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: charles River Laboratories, L'Arbresle, France.
- Age at study initiation: 8 week old (on the day of treatment)
- Weight at study initiation: 241+/-12 g (males), 227+/-9g (females)
- Fasting period before study: no data
- Housing: individually in polycarbonate cages with stainless steel lid (35.5 cm x 23.5 cm x 19.3 cm).
- Diet (e.g. ad libitum): free access to A04 C pelleted diet (UAR Villoisson, france)
- Water (e.g. ad libitum): filtered water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%):30 to 70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
On the day before treatment, the dorsal area of each animal was clipped (area = 6 cm x 8 cm) using an electric clipper. Only animals with healthy intact skin were used for the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
On removal of the dressing, any residual test substance was removed using a moistened cotton pad.

TEST MATERIAL
A single dose of 2000 mg/kg of DOTG in its original form was placed on a hydrophylic gauze pad pre-moistened with 2 mL of water and then applied to an area of the skin representing approximately 10 % of the total body surface of the animals.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Observations and weighing: The animals were observed frequently during the hours following administration of the test substance, for detection of possible treatment-related clinical signs. The animals were weighed individually just before administration of DOTG on day 1 and then on days 8 and 15.
- Necropsy of survivors performed: yes

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No deaths occurred during the study.

Clinical signs:

other: No clinical signs and no cutaneous reactions were observed during the study. A slight white coloration of the skin was noted in all animals on days 2 and 3.

Gross pathology:

Macroscopic examination of the main organs of the animals revealed no apparent abnormalities.

Other findings:

no

Table 1: Individual and mean body weight and weekly body weight change (g)

 

Dose mg/kg	sex	animals	Days
			1	1-8	8	8-15	15
2000	male	01	262	49	311	50	361
		02	243	47	290	48	338
		03	233	53	286	44	330
		04	236	49	285	52	337
		05	232	33	265	33	298
		M	241	46	287	45	333
		SD	12	8	16	8	23
2000	female	06	232	25	257	17	274
		07	226	12	238	3	241
		08	219	7	226	7	233
		09	218	25	243	17	260
		10	238	6	244	14	258
		M	227	15	242	12	253
		SD	9	9	11	6	16

 

1-8: body weight gain between D1 and D8

8-15: body weight gain between D8 and D15

M: mean

SD: standard deviation

Interpretation of results:

GHS criteria not met

Conclusions:

Under these experimental conditions, the dermal LD0 of the test substance DOTG is equal to or higher than 2000 mg/kg in rats, because no signs of toxicity were observed at this dose.

Executive summary:

The test substance was applied in its original form at the dose of 2000 mg/kg to the skin of one group of ten rats (5 males and 5 females). The test site was covered by a semi-occlusive dressing for 24 hours. Clinical signs, mortality and body weight gain were checked for a period of 14 days following the single application of the test substance. All animals were subjected to necropsy.

No clinical signs and no deaths were observed during the study. The body weight gain of the animals was similar to that of historical control animals. A slight white coloration of the skin was noted in all animals on days 2 and 3. No cutaneous reactions were observed. No apparent abnormalities were observed at necropsy in any animal.

Under these experimental conditions, the dermal LD0 of the test substance DOTG is equal to or higher than 2000 mg/kg in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD0

Value:

2 000 mg/kg bw

Quality of whole database:

The dermal acute toxicity study is reliable with a klimisch score of 1 and was performed according to the OECD 402 guideline and GLP compliance.

Additional information

Acute oral :

In the key study, a single oral administration of doses of 0 (control), 15, 30, 60 and 120 mg/kg of DOTG was performed on five male and five female rats in each group to study the acute toxic symptoms and the lethal dose. There are no mortality at 15 and 30 mg/kg bw and in control group.

Deaths occurred in the males and females in the 60 mg/kg (1 male and 3 females) and 120 mg/kg groups (4 males and 5 females). Treatment-related clinical signs were noted as follows: mydriasis, decrease in locomotor activity, bradypnea and tremors. Among males in the 60 mg/kg group, a trend of low values for body weights was noted on day 1 after administration and throughout the observation period in females in the 60 mg/kg group.The oral LD50 values are 85.3 mg/kg bw in males and 56 mg/kg bw in females.

In the first supporting study, groups of 10 rats (5 males/5 females) were exposed to DOTG at 103, 125, 150, 180 and 216 mg/kg bw by gavage.

Within a few hours after treatment all rats showed sluggishness and humpback behaviour. Several animals also showed tremors and convulsions. Mortality occurred from 90 minutes to 21 hours after treatment in all groups. Thereafter the survivors recovered and looked healthy again at the end of the observation period.The oral LD50 of DOTG was found to be 134 mg/kg bw in rats.

Acute dermal :

DOTG was applied in its original form at the dose of 2000 mg/kg to the skin of one group of ten rats (5 males and 5 females). The test site was covered by a semi-occlusive dressing for 24 hours. Clinical signs, mortality and body weight gain were checked for a period of 14 days following the single application of the test substance. All animals were subjected to necropsy.

No clinical signs and no deaths were observed during the study. The body weight gain of the animals was similar to that of historical control animals. A slight white coloration of the skin was noted in all animals on days 2 and 3. No cutaneous reactions were observed. No apparent abnormalities were observed at necropsy in any animal.

Under these experimental conditions, the dermal LD0 of the test substance DOTG is equal to or higher than 2000 mg/kg in rats.

Justification for classification or non-classification

Based on the available data, DOTG should be classified as Toxic if swallowed (Acute tox 3, H301) according to the Regulation EC N°1272/2008.

Justification: LD50(oral) is between 50 and 300 mg/kg bw.