N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-18 to 2018-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted 17 December, 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

30 May, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

December 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

EC Number:

205-181-1

EC Name:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Cas Number:

135-16-0

Molecular formula:

C19H23N7O6

IUPAC Name:

(2S)-2-[(4-{[(2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Step 1: animals no. 1-3: 148 – 164 g; Step 2: animals no. 4-6: 153 – 166 g
- Fasting period before study: Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted).
- Housing: The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Ad libitum, tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL


MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg


CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: It is the principle of the acute toxic class method that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test item to enable its classification. The item is tested using a stepwise procedure with up to four fixed doses. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 per step

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.
- Other examinations performed: clinical signs, body weight

Results and discussion

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

5 000 mg/kg bw

Based on:

test mat.

Effect levels 2

Key result

Sex:

female

Dose descriptor:

LD50

Remarks:

experimental

Effect level:

> 2 000

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

no mortality occurred

Clinical signs:

other: The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection, hunched posture and half eyelid-closure. All symptoms recovered within up to 2 days post-dose.

Gross pathology:

At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

Any other information on results incl. tables

Table 1: Clinical signs - Individual data (Step 1)

Step	Animal No. / Sex	Starting Dose (mg/kg bw)	Timepoint	Observations
1	1 / Female	2000	0 – 30 min	nsf
			30 – 60 min	Slightly reduced spontaneous activity, slight piloerection, half eyelid-closure
			60 – 240 min	Slightly reduced spontaneous activity, slight piloerection, hunched posture
			240 min - d 2	Slightly reduced spontaneous activity, slight piloerection
			d 2 – d 15	nsf
			0 – 30 min	nsf
	2 / Female		30 – 60 min	Slightly reduced spontaneous activity, slight piloerection, half eyelid-closure
			60 – 240 min	Slightly reduced spontaneous activity, slight piloerection, hunched posture
			240 min - d 2	Slightly reduced spontaneous activity, slight piloerection
			d 2 – d 15	nsf
			0 – 30 min	nsf
	3 / Female		30 – 60 min	Slightly reduced spontaneous activity, moderate piloerection, half eyelid-closure
			60 – 120 min	Slightly reduced spontaneous activity, hunched posture, moderate piloerection, half eyelid-closure
			120 – 240 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			240 min – d 2	Slightly reduced spontaneous activity, slight piloerection
			d 2 – d 15	nsf

Table 2: Clinical signs - Individual data (Step 2)

Step	Animal No. / Sex	Starting Dose (mg/kg bw)	Timepoint	Observations
2	4 / Female	2000	0 – 60 min	nsf
			60 – 180 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			180 - 240 min	Slight piloerection, hunched posture
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 – d 15	nsf
	5 / Female		0 – 60 min	nsf
			60 – 180 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			180 – 240 min	Hunched posture, slight piloerection
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 – d 15	nsf
	6 / Female		0 – 60 min	nsf
			60 – 180 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			180 – 240 min	Hunched posture, slight piloerection
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 – d 15	nsf

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single oral application of the test item Tetrahydrofolic acid (THFA) to rats at a dose of 2000 mg/kg body weight was associated with signs of toxicity but no mortality.
The median lethal dose of Tetrahydrofolic acid (THFA) after a single oral administration to female rats, observed over a period of 14 days is:
LD50 cut-off (rat): 5000 mg/ kg bw
Thus, the substance does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Calssification and Labelling of Chemicals (GHS).