Metformin hydrochloride

Endpoint summary

Description of key information

Two in vivo studies in rabbits are available for the evaluation of the skin irritating potential of Metformin hydrochloride. In one published study, undiluted Metformin was investigated, whereas in another study gel formulations consisting up to 2% metformin have been investigated. According to the results of these studies, Metformin hydrochlorid is considered to be not irritating to the skin

In a published in vivo study in rabbits, neat Metformin hydrochloride was irritating to the skin. In another study, no eye irritating potential was observed in rabbits treated with gel formulations consisting up to 2% metformin. Although the published study is of lower quality (e.g. individual values are not provided), an eye irritating potential of pure metformin hydrochlorid cannot be excluded. .

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981-1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Individual scores are not provided

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1979

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: J. Officiel de la Republique Francaise (1971). Methodes officielles d’analyse des cosmetiques et produits de beaute. Annexe II: Methode officielle pour la determination de i’irritation oculaire. Arrete du 16 Avril 1973. Journal Officiel 21 April. p. 3953.

Version / remarks:

1973

Qualifier:

according to guideline

Guideline:

other: Association Francaise de Normalisation (1982). Evaluation de lrritation et/au de la Corrosion Cutanie chez le Lapin. NF T03-263

Version / remarks:

1982

Principles of method if other than guideline:

The study compared the results of three different in vivo test methods for the investigation of skin irritation in rabbits: (i) the OECD method (used for this endpoint study record), (ii) a method published by French authorities for the testing of cosmetics and toiletries and (iii) a method proposed by AFNOR (Association Francaise de Normalisation).

GLP compliance:

no

Specific details on test material used for the study:

Beige Powder
Purity: no data
Storage: no data
Expiration: no data

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Roucher SA, Couhé.
- Age at study initiation: no data
- Weight at study initiation: 2.5 kg.
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

0.5 g of moistended test material

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48, and 72 hours and after 7 days and 14 days

Number of animals:

6 animals for each protocol (OECD, Cosmetics, AFNOR)

Details on study design:

TEST SITE
- Area of exposure: 6 cm x cm
- % coverage: 100
- Type of wrap if used: gauze pad (semiocclusive) or occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) 1, 24, 48, and 72 hours and after 7 days and 14 days

SCORING SYSTEM:
- Method of calculation:cf. Draize, Woodard & Calvery (1948)


Criteria used for interpretation based on Primary Dermal Irritation Index (PDII) according to AFNOR scale:
PDII below 0.5.................non irritant
0.5-3................................slightly irritant
3-5...................................moderately irritant
5-8...................................severely irritant

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0.13

Max. score:

8

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Remarks:

Individual scores for each animal are not provided

Irritant / corrosive response data:

Non irritant based on OECD protocol

Interpretation of results:

GHS criteria not met

Conclusions:

The test material is not irritant to the skin of rabbits in this semi-occlusive istudy when tested according to the OECD protocol.

Executive summary:

In a non-GLP primary dermal irritation study (Guillot and al, 1982) performed similarly to the OECD No. 404, six male New Zealand White rabbits were dermally exposed to 0.5 g of undiluted Metformin HCl to the dorsal surface of the trunk (skin was clipped before application) for 4 hours under an semiocclusive conditions. Skin reactions were assessed 1, 24, 48 and 72 hours after patch removal using the Draize scale. The irritancy of the test item was then defined on the basis of the the primary cutaneous index (PCI, comparable to the Primary Dermal Irritation Index PDII) using the scale appropriate for AFNOR.

The PCI/PDII is 0.13 (OECD protocol, maximum possible score = 8), the test substance was therefore considered as non-irritant to the skin.

Under the conditions of this test, no classification for skin irritation is required according to the criteria of the CLP Regulation (EC) N°1272/2008 and the Directive 67/548/EEC.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002-10-21 to 2003-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

yes

Remarks:

Exposure under occlusive conditions for 24 hours

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Formulations consisting of either 0.1% or 2% Metformin hydrochloride have been tested. In addition, a Placebo formulation was tested.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Albino rabbit, Crl:KBL(NZW)BR, female (f)
- Source: Charles River Wiga GmbH, Kißlegg
- Age at study initiation: approx. 18 weeks
- Weight at study initiation: 3.96 kg (3.50 - 4.95 kg)
- Housing: Becker, type K99/30 KU, floor area approx. 5400 cm2
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 22°C
- Humidity: 42 - 71%
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: From: 2002-11-14 To: 2002-11-21

Type of coverage:

occlusive

Preparation of test site:

shaved

Remarks:

The formulation was tested on intact and scarified skin. Only the results from intact skin are reported.

Vehicle:

other: gel formulation

Controls:

yes, concurrent vehicle

Amount / concentration applied:

0,5 ml

Duration of treatment / exposure:

24 hours

Observation period:

3, 24, 48, 72 hours after removal of patches up to 6 days after removal of patches.

Number of animals:

9 rabbits:
Metformin Topic Gel, Placebo: 3 animals
Metformin Topic Gel, 0.1% Metformin: 3 animals
Metformin Topic Gel, 2.0% Metformin: 3 animals

Details on study design:

Identification and adaptation
Healthy rabbits were allocated to the study group. The animals were kept in the experimental room for more than 7 days to allow them to acclimatize. Each animal was uniquely identified by a tattoo in the ear displaying the animal number. Individual cage cards were affixed to each cage displaying the study number, test material, day of treatment, and animal number.

Assignment
Female rabbits were used for this study. The numbers of the animals an the mean initial body weight at the start of the study are given below:
Test material formulation Animal number Mean initial body weight
Metformin Topic Gel, Placebo 19, 21, 30 3.90 (3.67 - 4.19) kg
Metformin Topic Gel, 0.1 % Metformin 23, 24, 27 4.00 (3.50 - 4.95) kg
Metformin Topic Gel, 2.0 % Metformin 20, 28, 29 3.99 (3.77 - 4.34) kg

Housing and diet
All rabbits were housed in an air-conditioned room of about 50 m2. A timer to provide a 12-hour light and a 12-hour dark regime controlled lighting.
Animals were kept separately in special rabbit cages (manufacturer: Becker; type K99/30 KU, floor area about 5400 cm2, a shelter with an integrated sitting board of about 1820 cm2; overall height: 60 cm) with plastic grids placed on mobile racks. The collection pans underneath the cages were cleaned at least three times per week. The cages were cleaned before the start of the study.
Temperature and relative humidity were measured using a thermohygrograph. The room temperature varied from 18 to 19°C and the relative humidity varied from 41 to 50%.
The rabbits received a commercial diet for rabbits, Provimi Kliba 3418.0 ad libitum and ssniff K snack, as well as fresh community tap water from Makrolon drinking bottles ad libitum.
According to the specifications given by the manufacturer, the diet had been checked by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics.
The drinking water was periodically analyzed according to the German regulations for human drinking water.

Grading scale for evaluation (according to OECD Guideline):

Erythema and eschar formation
Scores
--------------------------------------------------
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar
formation preventing grading of erythema 4
--------------------------------------------------
Maximum possible: 4
--------------------------------------------------

Edema formation
Scores
--------------------------------------------------
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well
defined by definite raising) 2
Moderate edema (raised approx. 1 mm) 3
Severe edema (raised more than 1 mm
and extending beyond area of exposure) 4
--------------------------------------------------
Maximum possible: 4
--------------------------------------------------

Total possible irritation score (maximum): 8

Mean score = Mean grading for erythema or edema of all the rabbits per time point
Mean cumulative score = Mean grading of all readings for erythema and edema per time point
Mean score per animal = Mean grading for each animal of erythema or edema (24, 48, and 72 hours after removal of the patches)

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

Placebo

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #4

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #4

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #5

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #5

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #6

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 1 day

Irritation parameter:

edema score

Basis:

animal #6

Remarks:

0.1% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #7

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #7

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #8

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #8

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #9

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 3 days

Irritation parameter:

edema score

Basis:

animal: #9

Remarks:

2.0% Metformin

Time point:

24/48/72 h

Score:

0

Max. score:

4

Other effects:

No signs of clinical toxicity were detected.
All animals survived the observation period.
Body weight development of the treated rabbits was inconspicuous.

Pathology

Macroscopic examination: No specific changes were seen in the samples of the intact skin.

Histopathological examination of the intact skin sites:  No specific changes were seen in the samples of the intact skin.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, a gel formulation consisting up to 2% Metformin is well tolerated, when applied to the skin of rabbits for 24 hours under occlusive conditions. A skin irritating potential of neat Metformin hydrochloride is unlikely According to the EEC-Directive 2001/59 the test material should not be classified as a skin irritant.

Executive summary:

Study Design
Metformin Topic Gel formulations (placebo,0.1%and 2.0 % formulation) were tested for for primary skin irritation. Therefore, 0.5 ml of the test material was spread onto 6cm² patches and applied to the intact skin of previously shaven rabbits for a 24 hours period under occlusive conditions. The first examination of the treated skin sites followed 3 hours after removal of the patches.Thereafter,examinations were performed daily for a further 6 days.

Results

Metformin Topic Gel Placebo: After exposure of Placebo formulation to the intact skin, no local irritation was seen

Metformin Topic Gel, 0.1% Metformin: After exposure to intact skin, one animal showed erythema (score: 1) for 3 to 24 hours

Metformin Topic Gel, 2.0% Metformin: After exposure to intact skin, one animal showed erythema (score: 1 and 2) for 3 to 48 hours

Pathology: All test sites were examined pathologically. The application sites (intact skin) of all dose groups were examined macroscopically. There were no findings,that could be assigned to the test material formulations 0.1% and 2.0% Metformin Topic Gel.

Conclusion

Based on the results of this study, the skin irritating potential of the active substance Metformin is considered to be low.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Individual scores are not provided

Justification for type of information:

Comperative study using the AFNOR protocol in comparison to the OECD protocol.

References:
French authorities for the testing of cosmetics and toiletries (Journal Officiel de la Republique Francaise, 1971 & 1973
Association Francaise de Normalisation (1982). Evaluation de I’lrritation et/au de la Corrosion Oculaires, chez le Lapin. AFNOR (Project): TD3-B dot. 37.
Organisation for Economic Co-operation and Development (1979). OECD Short-term and Long-term Toxicology Groups, Final Report: Acute Eye Irritation/Corrosivity. 31 December, p. 40.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1979

Qualifier:

according to guideline

Guideline:

other: Association Francaise de Normalisation (1982). Evaluation de I’lrritation et/au de la Corrosion Oculaires, chez le Lapin. AFNOR (Project): TD3-B doc. 37.

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

other: J. Officiel de la Republique Francaise (1971). Methodes officielles d’analyse des cosmetiques et produits de beaute. Annexe II: Methode officielle pour la determination de i’irritation oculaire. Arrete du 5 Avril 1971. Journal Officiel 21 April. p. 3862.

Version / remarks:

1971

Qualifier:

according to guideline

Guideline:

other: J. Officiel de la Republique Francaise (1973). Methodes officielles d’analyse des cosmetiques et produits de beaute. Annexe II: Methode officielle pour la determination de i’irritation oculaire. Arrete du 16 Avril 1973. Journal Officiel 05 June. p. 3953.

Version / remarks:

1973

Principles of method if other than guideline:

The scoring system of Kay and Calandra (1962, J. Soc. cosmet. Chem. 13, pp. 281) was used.

GLP compliance:

no

Specific details on test material used for the study:

Beige Powder
pH 6-7 (saturated aqueous solution)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Roucher SA, Couhe, France
- Age at study initiation:
- Weight at study initiation: 2 - 5 kg
- Housing: group
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

Vehicle:

unchanged (no vehicle)

Controls:

other: comperative study including 56 compounds with also non irritant compounds to be used as control

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

rinsing after:
a) 4s
b) 30 s
c) no rinsing performed

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

18 males:
first group of 6 males without rinsing, second group of 6 males with rinsing after 4s, and third group of 6 males with rinsing after 30s.

Details on study design:

The test substance was ground, to produce a fine dust for instillation. This involved a deviation from the Cosmetic protocol,
which specifies 20% (w/v) aqueous dilution of powders. The pH of the test substance was determined with 7.6

Rinsing solution Rinsing, when required, was generally carried out with Dacryoserum (from Chibret Laboratories, Paris), a solution of boric acid (1.8 g), sodium borate (1.2 g), sodium chloride (0.3 g) and phenylmercury
borate (0.002 g) in 100 ml rose distilled water QSP.
Dosing and observation. Test substance (100 mg) was instilled into the lower conjunctival cul-de-sac of one eye of each rabbit; the other eye constituted the control.

Interpretation
At each observation time, lesions of the conjunctiva. iris and cornea were scored separately using a numerical system based on that of Kay & Calandra (1962), the sum of these scores being the individual
ocular irritation index (IOI) for each animal. Calculation of the mean (and standard deviation) of the IOI values obtained at any one time gave the mean ocular index (MOI). The greatest MOI obtained over the range of observation times was identified as the acute ocular irritation index (AOI), which was used for the preliminary classification of a substance in one of six categories between ‘non-irritant’ and ‘extremely
irritant’. In the AFNOR interpretation, which takes into account not only the maximum intensity of the lesion but also its reversibility, the preliminary classification is modified if recovery is not virtually complete within 4 days. by evaluating the fall in MOI with time and, after 7 days, the frequency of the persisting lesions.

Irritation parameter:

other: Acute Ocular Irritation Index (AOI)

Basis:

mean

Time point:

other: 1 hour

Score:

16.67

Max. score:

110

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: MOI = 4.17 (day 4)

Remarks:

no rinsing

Irritation parameter:

other: Acute Ocular Irritation Index (AOI)

Basis:

mean

Time point:

other: 1 hour

Score:

16.83

Max. score:

110

Reversibility:

not fully reversible within: 4 days

Remarks on result:

other: MOI = 0 (day 4)

Remarks:

rinsing after 30 s

Irritation parameter:

other: Acute Ocular Irritation Index (AOI)

Basis:

mean

Time point:

other: 1 hour

Score:

9.33

Max. score:

110

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: MOI = 0.83 (day 4)

Remarks:

rinsing after 4 s

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, based on these results, Metformin hydrochloride is considered as irritating to eyes and classified into category 2, H319 according to the CLP Regulation (EC) N° 1272/2008 and according to the Directive 67/548/EEC.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-10-21 to 2003-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

29 December 1992

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Formulations consisting of either 0.1% or 2% Metformin hydrochloride have been tested. In addition, a Placebo formulation was tested.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Strain: Albino rabbit, Crl:KBL(NZW)BR, female (f)
- Source: Charles River Wiga GmbH, Kißlegg
- Age at study initiation: approx. 15 weeks
- Weight at study initiation: 3.24 kg (mean)
- Housing: Becker, type K99/30 KU, floor area approx. 5400 cm2
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 20°C
- Humidity: 42 - 71%
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: From: 2002-10-22 To: 2002-10-30

Vehicle:

other: The test item is a formulation

Controls:

yes, concurrent vehicle

Amount / concentration applied:

0,1 ml

Duration of treatment / exposure:

0.1 mL test material was instilled into the conjunctival sac of the left eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test material was placed. The right eye remained untreated and served as control. After instillation, the eyelids were closed for about 30 seconds.

Observation period (in vivo):

The rabbits were examined for eye irritation and for changes in behavior and general condition 1 hour after treatment, after 24, 48, or 72 hours, then daily up to day 8 of the experimental part.

Number of animals or in vitro replicates:

9 rabbits:
Metformin Topic Gel, Placebo: 3 animals
Metformin Topic Gel, 0.1% Metformin: 3 animals
Metformin Topic Gel, 2.0% Metformin: 3 animals

Details on study design:

-- Identification and adaptation
Healthy rabbits were allocated to the study group. The animals were kept in the experimental room for more than 7 days to allow them to acclimatize.
Each animal was uniquely identified by a tattoo in the ear displaying the animal number. Individual cage cards were affixed to each cage displaying the study number, test material, day of treatment, and animal number.

-- Assignment
3 female rabbits per formulation, a total of 9 rabbits, were used for this study. The mean initial body weight at the start of the experimental part was 3.24 kg.

-- Housing and diet
All rabbits were housed in an air-conditioned room of about 50 m2. Lighting was controlled by a timer to provide a 12-hour light and a 12-hour dark regime.
They were kept separately in special rabbit cages (manufacturer: Becker; type K99/30 KU, floor area about 5400 cm2, a shelter with an integrated sitting board of about 1820 cm2; overall height: 60 cm) with plastic grids placed on mobile racks. The collection pans underneath the cages were cleaned at Ieast three times per week. The cages were also cleaned before the start of the experimental part.
Temperature and relative humidity were measured using a thermohygrograph. The room temperature varied from 19 to 20 °C and the relative humidity varied from 42 to 71%.
The rabbits received a commercial diet for rabbits, Provimi Kliba 3418.0 ad libitum and ssniff K snack, as well as fresh community tap water from Makrolon drinking bottles ad libitum.
According to the specifications given by manufacturer, the diet had been checked by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics.
The drinking water was periodically analyzed according to the German regulations for human drinking water.

-- Preparation
The test material was administered undiluted.

–- Administration and dose level
To ensure that only rabbits with normal eyes were included in the study, approximately 24 hours before treatment ophthalmologic examinations were performed after instillation of a 0.15 % fluorescein solution (Dr. Thilo & Co.) using an ophthalmoscope. Animals with eye defects, injury or irritation were excluded.
0.1 mL test material, was instilled into the conjunctival sac of the left eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test material was placed. The right eye remained untreated and served as control. After instillation, the eyelids were closed for about 30 seconds.
The study consisted of an initial test with one rabbit and a confirmatory test with two further animals.

-- Observation for clinical symptoms
The rabbits were examined for eye irritation and for changes in behavior and general condition 1 hour after treatment, after 24, 48, or 72 hours, then daily up to day 8 of the experimental part.

Eye changes were evaluated according to DRAIZE, OECD and EEC recommendations.


-- Evaluation of eye reactions

- Cornea Scores
A) Opacity-degree of density (area most dense taken for reading)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling
of normal lustre), details of iris clearly visible 1
Easily discernible translucent area, details of iris slightly obscured 2
Nacrous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

B) Area of cornea involved not evaluated (Rating according to the DRAIZE method)

- Iris
A) Normal 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal
hyperemia, or injection, any of these or combination of any thereof,
iris still reacting to light (sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2

- Conjunctivae
A) Redness (refers to palpebral and bulbar conjunctivae cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2
Diffuse beefy red 3

B) Chemosis: lids and/or nictating membranes
No swelling 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4

C) Discharge (Rating according to the DRAIZE method )
No discharge 0
Any amount different from normal (does not include small
amounts observed in inner canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adjacent to lids 2
Discharge with moistening of the lids and hairs,
and of a considerable area around the eye 3

Mean score of all animals = Mean grading for irritations of cornea, iris, and conjunctivae per time point
Mean score per animal = Mean grading for each animal of irritations of cornea, iris, and conjunctivae (1, 24, 48, and 72 hours after application)
Maximum value = Maximum grading of a sign of irritation within a period

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No signs of irritation could be observed at the cornea, iris or conjunctivae.

Other effects:

No signs of clinical toxicity were detected.
All animals survived the observation period.
Body weight development of the treated rabbits was inconspicuous.

Interpretation of results:

GHS criteria not met

Conclusions:

A gel formulation consisting up to 2% Metformin was not irritating to the eyes of rabbits.

Executive summary:

Study Design

Metformin Topic Gel. two Metformin concentrations (0.1% and 2.0%) and the placebo formulation, were tested for its eye irritating potential. For the test of primary eye irritation, 0.1 mL of each formulation was applied into the conjunctival sac of rabbits. The study was started as an initial test with 1 animal and followed by the confirmatory test with further 2 animals per formulation.

Results

No signs of irritation were observed at cornea, iris, or conjunctivae in rabbits treated Metformin Topic Gel placebo, 0.1% Metformin and 2.0% Metformin. The individual scores for all animals were 0 for all formulations at any time point.

Conclusion

Gel formulations consisting up to 2.0% Metformin were not irritating to the eyes of rabbits.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vivo:

For this endpoint data from a publication are available (Guillot and al, 1982). The study was performed according to three methods (OECD, AFNOR, Cosmetic and toiletries).

The test performed according to OECD method, was selected as the key study with reliability 2.

The other two tests were considered as supporting data.

Groups of 6 male New Zealand White rabbits were dermally exposed to 0.5 mL of undiluted metformin HCl to the dorsal surface of the trunk (skin was clipped (OECD method) and clipped + scarified (AFNOR + Cosmetic methods) before application) ) for 4 hours (OECD and AFNOR) and for 23 hours (Cosmetic) under an occlusive dressing. Skin reactions were assessed 1, 48 and 1, 24, 48 and 1, 24, 48, 72 hours after patch removal for Cosmetic, AFNOR and OECD respectively using the Draize scale. The irritancy of the test item was then defined on the basis of the primary dermal irritation index (PDII) using the AFNOR and Cosmetic scale.

The PCI is 0.13 (OECD protocol), the test substance was therefore considered as non-irritant to the skin.

Under the conditions of this test, no classification for skin irritation is required according to the criteria of the CLP Regulation (EC) N°1272/2008 and the Directive 67/548/EEC.

Eye irritation in vivo:

For this endpoint data from a publication are available (Guillot and al, 1982) with reliability 2. This study is considered as the key study despite the limited level of details.

In a primary eye irritation study (J.P. Guillot and al, 1982), using a procedure that complied with the main requirement of three methods (OECD, AFNOR, Cosmetic and toileteries), 0.1 mL of Metformin HCl was instilled into the conjunctival sac of the eye of treated groups of 6 New Zealand white rabbits (males) (eyes washed after 4 and 30 sec and unwashed). Scoring was assessed at 1 hr and 1, 2, 3, 4, 7 days post instillation. Irritation was scored by the method according AFNOR.

- As the Acute Ocular irritation Index (AOI) = 16.67 and the Mean Ocular irritation Index (MOI) = 4.17 (Day 4), Metformin HCl was considered as irritating to rabbits on ocular application without rinsing according to AFNOR scale.

- As the Acute Ocular irritation Index (AOI) = 16.83 and the Mean Ocular irritation Index (MOI) = 0 (Day 4), Metformin HCl was considered as irritating to rabbits on ocular application when rinsed after 30 sec according to AFNOR scale.

- As the Acute Ocular irritation Index (AOI) = 9.33 and the Mean Ocular irritation Index (MOI) = 0.83 (Day 4), Metformin HCl was considered as slightly irritating to rabbits on ocular application when rinsed after 4 sec according to AFNOR scale.

In conclusion, based on these results, Metformin HCl is considered as irritating to eyes and classified into category 2, H319 according to the CLP Regulation (EC) N° 1272/2008 and according to the Directive 67/548/EEC.

Justification for classification or non-classification

Metformin HCl is considered as irritating to eyes and classified into category 2, H319 according to the CLP Regulation (EC) N° 1272/2008 and according to the Directive 67/548/EEC.

There is no classification for skin irritation is required according to the criteria of the CLP Regulation (EC) N°1272/2008 and the Directive 67/548/EEC.