(2S)-5-amino-2-[(aminoacetyl)amino]-5-oxopentanoic acid

Endpoint summary

Description of key information

Oral (OECD 401), rat, LD50 (m, f) > 5110 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 4879 mg/kg bw anhydrous glycyl-L-glutamine

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-11-19 to 1992-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 24 February, 1987

Deviations:

yes

Remarks:

only 2 dose levels but dose levels are sufficient for evaluation, maximum dose volume applied was 21.5 mL/kg bw

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

other: Bor: WISW (SPFCpb)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co. KG, Borchen, Germany
- Age at study initiation: males 8 weeks; females 9 weeks
- Weight at study initiation: males 150 - 164 g; females 138 - 146 g
- Fasting period before study: 16 hours before treatment
- Housing: individually in Macrolon cages type II
- Diet: standard diet, ad libitum (ssniff R, special diet for rats)
- Water: drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 21.6
- Humidity (%): 40 - 69
- Photoperiod (per hr): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (w/v)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 110 mg/mL (2370 mg/kg bw dose) and 237 mg/mL (5110 mg/kg bw dose)
- Amount of vehicle (if gavage): 21.5 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 21.5 mL/kg bw

DOSAGE PREPARATION : The test item was suspended in the vehicle immediately before dosing, using a homogenizer and by shaking.

Doses:

2370 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 2177 mg/kg bw anhydrous glycyl-L-glutamine
5311 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 4879 mg/kg bw anhydrous glycyl-L-glutamine

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: at 4 to 6 h after dosing and once daily thereafter
- Frequency of weighing: day 0 (prior to dosing), thereafter at weekly intervals up to the end of the study
- Necropsy of survivors performed: yes
- Other examinations performed: macroscopical examination included external appearance, body orifices, thoracic and abdominal cavities. Histopathology was carried out on the testes and epididymes of males, and the ovaries of females.

Statistics:

no

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 110 mg/kg bw

Based on:

test mat.

Remarks:

test item glycyl-L-glutamine monohydrate

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 4 879 mg/kg bw

Based on:

other: anhydrous glycyl-L-glutamine

Mortality:

2370 mg/kg bw: no deaths
5110 mg/kg bw: 0/5 males and 2/5 females died 3 h post-application.

Clinical signs:

other: 2370 mg/kg bw: slight hypokinesia and decrease of muscle tone was observed in 1/5 males and 2/5 females. 5110 mg/kg bw: slight to severe hypokinesia, slight clonic convulsions, decrease of muscle tone, and loss of righting reflexes, restrained gait, piloe

Gross pathology:

2370 mg/kg bw: In 2/5 male animals the testes appeared reduced in size.
5110 mg/kg bw: In 1/5 male animals the testes appeared reduced in size. A reddened lung was noted in the 2 deceased females.
The observations were considered toxicologically relevant and attributed to treatment.

Other findings:

Testes:
The testes were affected by focal and/or diffuse atrophy of the seminiferous epithelium. In minimal to moderate cases degenerative changes were observed in spermatocytes and spermatids, resulting in a reduction or absence of these cells in specific stages of the seminiferous epithelium. Spermatocytes were predominantly affected in the zygotene stage of the meiotic cell division. Degenerative changes and a delay in the maturation of spermatids occurred in the first five stages of the cycle of the seminiferous epithelium. Affected tubules often had multinucleated giant cells. In marked to massive cases the epithelium of the seminiferous tubules consisted only of spermatogonia and Sertoli-cells or was characterized by the Sertoli-only change.
Diffuse atrophy of the seminiferous epithelium occured in 2/5 rats treated with 5110 mg/kg bw and in 5/5 rats treated with 2370 mg/kg bw. The finding was graded as marked to massive in animals of the high dose group and minimal to moderate in animals of the low dose group. Focal atrophy was observed in 1/5 and 3/5 animals treated with 5110 and 2370 mg/kg bw, respectively. It was graded slight to marked.

Epididymides:
The epididymides of animals of both dose groups had a treatment-related reduction of spermatosoma. This finding was associated with the occurrence of immature, multinucleated and degenerated speratids / spermatozoa. Four/5 and 5/5 animals treated with 5110 and 2370 mg/kg bw were affected by this change. It was graded slight to massive.

Ovaries:
The examined ovaries did not show any treatment-related findings.

Interpretation of results:

GHS criteria not met

Conclusions:

After oral application of the test substance the LD50 was above 5110 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 4879 mg/kg bw anhydrous glycyl-L-glutamine, for male and female rats.
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-02-16 to 1994-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 24 Feb 1987

Deviations:

yes

Remarks:

maximum dose volume applied was 21.5 mL/kg bw

Qualifier:

according to guideline

Guideline:

other: Comission Directive No. 91/507/EEC

Version / remarks:

adopted 19 July, 1991

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

other: Hsd/Win:WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Halle, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 158 - 210 g
- Housing: individually in Macrolon cages, type II
- Diet: standard diet, ad libitum (ssniff R, special diet for rats)
- Water: drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22
- Humidity (%): 60 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (w/v)

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
- Amount of vehicle (if gavage): 21.5 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 21.5 mL/kg bw

DOSAGE PREPARATION : The test item was suspended in the vehicle using a homogenizer and an ultrasonic bath

Doses:

237 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 217.7 mg/kg bw anhydrous glycyl-L-glutamine
750 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 688.9 mg/kg bw anhydrous glycyl-L-glutamine
2370 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 2177 mg/kg bw anhydrous glycyl-L-glutamine

No. of animals per sex per dose:

5 males (main group) and 6 males (satellite group)

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals of the main groups were continously observed for clinical symptoms for the first 4 to 6 hours after administration and then once daily; mortality was checked twice daily; body weight were recorded at the beginning, 7 days after administration and 14 days after administration.
- Necropsy of survivors performed: yes
- Other examinations performed: blood samples were collected from satellite animals prior to administration, 30 and 120 minutes post-administration from 3 rats/group and 10, 60 and 240 minutes post-administration from the remaining 2 rats/group. Plasma was isolated and levels of aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, proline, glycine, alanine, valine, glycyl-glutamine, methionine, cystine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, ornithine, lysine, histidine and arginine were quantified. In addition, organ weights from the testes incl. epididymides of all animals of the main group were collected and histopathology of testes and epididymides was performed.

Statistics:

Mean values of body weights, organ weights and standard deviations were calculated separately for each group. For statistical evaluation of body weights and organ weights the DUNNETT-Test was used.

Sex:

male

Dose descriptor:

LD50

Effect level:

> 2 370 mg/kg bw

Based on:

test mat.

Remarks:

test item glycyl-L-glutamine monohydrate

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 177 mg/kg bw

Based on:

other: anhydrous glycyl-L-glutamine

Mortality:

No mortality was observed up to the highest dose tested.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy and histopathological examination revealed no treatment-related findings.

Other findings:

Biochemical investigations:
The results of the analytical and biochemical investigations showed a dose-related increasing of the plasma levels of glycine, glutamine, glutamic acid, and alanine during the first 30 minutes after application. During the following 2 hours the levels decreased to the baseline values measured before administration.

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 for male rats was determined to be > 2370 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 2177 mg/kg bw anhydrous glycyl-L-glutamine, for male and female rats.
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies with the registered substance. The selected studies are thus sufficient to fulfil the standard information requirements of Regulation (EC) No. 1907/2006 (REACH).

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:

23 December 2021 - 26 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8-10 weeks old
- Weight at study initiation: 174 to 197 g
- Fasting period before study: no
- Housing: individually housed in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum
- Water: Municipal tap-water was freely available to each animal via water bottles
- Method of randomisation in assigning animals to test and control groups: all animals within ± 20% of the sex mean body weights

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C (target 20-24°C)
- Humidity (%): 50-60% (target 40-70%)
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 January 2022 To: 26 January 2022

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Remarks:

The samples were applied after moistening with 0.5. mL water (Elix), to ensure close contact to the skin.

Details on dermal exposure:

TEST SITE
- Area of exposure: 5 x 7 cm
- % coverage: 18 cm2 for females
- Type of wrap if used: surgical gauze patch (Surgy 1D), successively covered with Coban elastic bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with water
- Time after start of exposure: 24 hours

Duration of exposure:

24 hours

Doses:

2178 mg/kg bw (monohydrate form; test item; CAS 172669-64-6) equivalent to
2000 mg/kg bw (anhydrous form; CAS 13115-71-4)

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: post-dose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter; animals were weighed individually on Days 1 (pre-dose), 8 and 15
- Necropsy of survivors performed: yes
- Irritation: skin reactions were assessed at least once daily after removal of the dressing and test
item

Preliminary study:

A range finding study was performed in order to select the dose causing no mortality or significant toxicity to be used in the main study. One animal was dosed at 2000 mg/kg.

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

> 2 178 mg/kg bw

Based on:

test mat.

Remarks:

monohydrate form (CAS 172669-64-6; test item)

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Remarks:

anhydrous form (CAS 13115-71-4)

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

No mortality occurred.

Clinical signs:

other:

Body weight:

other body weight observations

Remarks:

The body weight gain shown by two of the animals during the observation period was within the range expected for rats used in this type of study. The lack of body weight gain between Days 1 and 15 in one animal was considered not indicative of toxicity, based on the absence of any corroborative findings in these animals.

Gross pathology:

No abnormalities were found at macroscopic postmortem examination of the animals.

Interpretation of results:

GHS criteria not met

Remarks:

Based on these results, Glycyl-L-Glutamine monohydrate does not have to be classified and has no obligatory labelling requirement for acute dermal toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Conclusions:

The dermal LD50 value of Glycyl-L-Glutamine monohydrate (CAS 172669-64-6) in Wistar Han rats was established to exceed 2178 mg/kg body weight, equivalent to 2000 mg/kg body weight of the anhydrous form Glycyl-L-Glutamine (CAS 13115-71-4).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study with the registered substance. The selected studies are thus sufficient to fulfil the standard information requirements of Regulation (EC) No. 1907/2006 (REACH).

Additional information

Two in vivo studies are available analysing the acute oral toxicity of the test item in rodents. 

The first study was conducted in Bor:WISW rats according to OECD guideline 401 and in compliance with GLP (92-0043-DNT). The test substance was dissolved in 0.5% carboxymethyl cellulose and administered as a single dose by oral gavage at doses of 2370 and 5110 mg/kg bw. Groups of 5 rats per sex received the test item or the corresponding vehicle and were observed for a period of 14 days following administration. Individual body weights were recorded prior to administration and at weekly intervals thereafter. At study termination, all animals were subjected to gross necropsy.

Mortality was observed in 2/5 females at 5110 mg/kg bw within 3 hours post-administration. Clinical signs of toxicity were observed in 1/5 males and 2/5 females at 2370 mg/kg bw and comprised slight to severe hypokinesia and decreased muscle tone. Slight to severe hypokenesia, slight clonic convulsions, loss of righting reflexes, restrained gait, piloerection, slight tremor, diarrhea and/or sunken sides was observed in all animals at 5110 mg/kg bw. The Symptoms were present from 29 minutes post dosing to 1 day post dosing or lasted until sacrifice. The observations were considered toxicologically relevant and attributed to treatment.

There was no effect on body weight or body weight gain. At gross necropsy, smaller testes were noted in 2/5 males at 2370 mg/kg bw and in 1/5 males at 5110 mg/kg bw, and reddening of the lung was observed in the two deceased females of the high-dose group. The histopathological examination showed diffuse atrophy of the seminiferous epithelium of the testes in 5/5 males of the low-dose group (minimal to moderate grade) and in 2/5 males of the high-dose group (massive grade). Focal atrophy of the seminiferous epithelium of the testes was noted to a slight degree in 3/5 males at 2370 mg/kg bw and in 1/5 males at 5110 mg/kg bw. In line with these findings, the epididymis of both dose groups showed a reduction in spermatozoa (5/5 males at 2370 mg/kg bw and 4/5 males at 5110 mg/kg bw). The finding was associated with the occurrence of immature, multinucleated and degenerated spermatids/spermatozoa, of slight to massive degree and attributed to the treatment.

Under the conditions of the study, the acute oral LD₅₀ value for male and female rats was > 5110 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 4879 mg/kg bw anhydrous glycyl-L-glutamine.

 

A second study on acute oral toxicity was conducted in male Hsd/Win:WU rats according to OECD guideline 401 and in compliance with GLP (94-0001-DNT). The test substance was dissolved in 0.5% carboxymethyl cellulose and administered as a single dose by oral gavage at doses of 237, 750 and 2370 mg/kg bw to groups of 5 male rats. A group of 5 males received the vehicle and served as control. In addition, a group of 6 satellite animals per dose was included, which was treated with the test item according to the same study protocol as for the main groups.

All the animals were observed for a period of 14 days post-administration, during which clinical signs of toxicity and mortality were recorded once or twice daily. Individual body weights were recorded prior to administration and at weekly intervals thereafter. In addition, plasma amino acid levels were quantified from satellite animals, approximately 30 minutes prior to administration and at 30 and 120 minutes from 3/5 satellite animals and 10, 60 and 240 minutes post-administration from 2/5 satellite animals. At scheduled necropsy, all animals underwent gross necropsy and histopathological examination.

There was no mortality and no symptoms of systemic toxicity were recorded. The body weight remained unaffected by treatment. The Gross necropsy and histopathology examination showed no treatment-related findings. A dose-related increase in the plasma amino acids was recorded during the first 30 minutes after application, followed by a decrease to baseline values within 2 hours post-administration.

Under the conditions of the test the acute oral LD₅₀ for male rats was >2370 mg/kg bw (test item glycyl-L-glutamine monohydrate), equivalent to 2177 mg/kg bw anhydrous glycyl-L-glutamine, for male and female rats.

 

One study was performed to determine the potential toxicity of Glycyl-L-Glutamine monohydrate , when given by a single dermal dose.

The study was carried out according to OECD TG 402 (2017). Initially, Glycyl-L-Glutamine monohydrate was administered to a single female Wistar Han rat by a single dermal application at 2000 mg/kg bw for 24 hours in a range finder study. Based on the results, the main study was performed by dosing two females at 2000 mg/kg bw. A factor 1.089 was used to correct for the purity of the test item and the corrected dose concentration was calculated to be 2178 mg/kg bw. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (Day 15).

No mortality occurred. No clinical signs of systemic toxicity and no irritation was noted. The body weight gain shown by two of the animals during the observation period was within the range expected for rats used in this type of study. The lack of body weight gain between Days 1 and 15 in one animal was considered not indicative of toxicity, based on the absence of any corroborative findings in these animals. No abnormalities were found at macroscopic postmortem examination of the animals.

The dermal LD₅₀ value of Glycyl-L-Glutamine monohydrate (CAS 172669-64-6) in Wistar Han rats was established to exceed 2178 mg/kg body weight, equivalent to 2000 mg/kg body weight of the anhydrous form Glycyl-L-Glutamine (CAS 13115-71-4).

Justification for classification or non-classification

The available data on acute oral toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.

No classification for acute oral toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.