Guanidinium nitrate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Screening study:

In accordance with column 2 of REACH Annex VIII the study on “Screening for reproductive/developmental toxicity, one species” (required in section 8.7.1) does not need to be conducted if a pre-natal developmental toxicity study is available.

Data from a developmental toxicity are available. Thus, conducting of a screening study for reproductive/ developmental toxicity is not necessary.

Fertility data from repeated dose toxicity:

In a sub-chronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), the read-across substance Guanidine hydrochloride was administered to 10 Wistar rats/sex/dose in water, by gavage at dose levels of 0, 50, 100 and 300 mg/kg bw/day.

In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects a satellite group of 5 rats/sex was exposed at dose levels of 0 and 300 mg/kg bw/day (control and HD).

To evaluate possible toxic effects on fertility, the estrous cycle was examined at defined time points of the treatment and recovery period and epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were evaluated at the end of the treatment and recovery period. Moreover, a detailed histopathological evaluation of the reproductive organs was performed.

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.

 

Justification for read-across:

Guanidine hydrochloride and guanidine nitrate dissociate in biological fluids to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the systemic intrinsic properties of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. Systemic effects of guanidine hydrochloride are expected to be based on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine hydrochloride for effects of the guanidine ion is considered valid. The effects of the nitrate ion will be discussed separately by read-across. This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies.

A more detailed justification is attached and outlined in CSR chapter 1.1.2 as well.

 

Effects of nitrate ions

Data are available from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 with Potassium nitrate. For the reproductive subgroup of the study 5 male and 10 female Sprangue-Dawley rats per dose were exposed at dose levels of 0, 250, 750 and 1500 mg/kg bw/day by gavage for a period of 28 (males) and approximately 53 (females) days.

There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring.

The NOAEL for reproductive toxicity was established at the highest tested dose at 1,500 mg/kg bw/day (equivalent to 920 mg/kg bw/day nitrate)

Further, the OECD SIAR, Nitrates Category, 2007 concluded that members of the nitrate category are not considered reproductive or developmental toxicants.

 

Conclusion:

No effect on reproductive toxicity was observed in the Screening study with Potassium nitrate and a NOAEL was 1500 mg/kg bw/day (equivalent to 920 mg/kg bw/day nitrate), confirmed by OECD SIAR, Nitrates Category, 2007.

Taken together and as no effects on fertility parameters were observed in the sub-chronic toxicity with Guanidine hydrochloride, the derived NOAEL of 300 mg/kg bw/day is considered a reliable dose descriptor for fertility of guanidine nitrate. In particular, considering that adjustment of molecular weight will lead to higher value for guanidine nitrate.

For assessment of Guanidine nitrate the NOAEL of 100 mg/kg bw/day obtained for repeated dose toxicity from the sub-chronic toxicity study is considered the reliable dose descriptor, the respective DNELs are also protective for reproductive toxicity.

 

2-generationen study:

The conduct of a Two-generation reproductive toxicity study is omitted according to Annex XI section 3.2 of REACH Regulation (EC) No 1907/2006 based on an exposure based adaptation.

The rules outline in section 3.2 (a) of Annex XI were used for exposure based adaptation:

An exposure assessment according to Annex I of REACH Regulation was conducted. This assessment is built on the use of exposure scenarios covering all relevant stages of the entire life cycle and respective exposure estimates, including a qualitative and quantitative risk characterisation.

All of the following conditions are fulfilled:

 

(i) the results of the exposure assessment covering all relevant exposures throughout the life cycle of the substance demonstrate the absence of or no significant exposure in all scenarios of the manufacture and all identified uses as referred to in Annex VI section 3.5

 

(ii) a DNEL or a PNEC can be derived from results of available test data for the substance concerned taking full account of the increased uncertainty resulting from the omission of the information requirement, and that DNEL or PNEC is relevant and appropriate both to the information requirement to be omitted and for risk assessment purposes

 

(iii) the comparison of the derived DNEL or PNEC with the results of the exposure assessment shows that exposures are always well below the derived DNEL or PNEC;

 

The full comprehensive exposure assessment is outlined in chapters 9 and 10 of the chemical safety report (CSR). The spectrum of identified applications is very small and specific. The identified applications are use as intermediate, use in fertilizers and explosives. The results of exposure and risk assessment covering all relevant exposures throughout the life cycle of the substance demonstrate a low exposure in all scenarios of manufacture, formulation, professional use, consumer use and indirect exposure of humans via the environment.

The very small spectrum of identified uses as referred to in Annex VI section 3.5 is documented in chapters 9 and 10 of CSR. Especially consumer use is limited to infrequent use in fertilizers at very low concentrations of 0.2 % w/w and at low volume. Same is true for "service live" of use in airbags,  where an exposure to the substance through the activated airbag is very rare, short time and only of very low concentration as the guanidine nitrate is consumed in the ignition.

 

Strictly conservative DNELs have been derived taking full account of the increased uncertainty resulting from the omission of the information requirement.

Results from the available test data include a pre-natal developmental toxicity study in rats and a sub-chronic toxicity study in rats. In addition to guideline requirements an assessment of fertility parameters was conducted within the sub-chronic toxicity study, especially to support the robustness of exposure based adaptation. No effects on estrous cycle, epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were observed.

These data are considered to be appropriate and reliable dose descriptors for DNEL derivation for omitting a prenatal developmental toxicity study in the second species and a two-generation reproductive toxicity study.

No substance-related adverse effects regarding reproductive/developmental endpoints were found in any of the tests conducted. Effects on general toxicity were observed in acute toxicity studies and in the sub-chronic toxicity study. As well in the developmental toxicity study maternal toxicity was observed. Guanidine Nitrate has no genotoxic properties as proven in the full genotoxic test battery.

The NOAELs used to derive the DNELs for fertility and developmental toxicity correspond to the maximum doses tested. The DNELs fertility have been derived from results of the sub-chronic repeated dose toxicity study, taking full account of the potential increased uncertainty resulting from the omission of the information requirement by applying an additional assessment factor of 2. This additional assessment factor has also been applied to DNELs derived for developmental toxicity.

The derived DNELs for fertility and developmental toxicity are all higher than the DNELs derived based on the NOAEL observed for systemic toxicity in the sub-chronic toxicity study.

Accordingly, systemic toxicity was considered the most sensitive Endpoint and thus the reliable dose descriptor for further assessment. The derived DNELs are protective for fertility and developmental toxicity including the potential increased uncertainty resulting from the omission of the information requirement and thus are relevant and appropriate for both the information requirement to be omitted and for risk assessment purposes.

No classification according to GHS Regulation EC No 1272/2008 for acute or chronic aquatic toxicity is required for guanidine nitrate, hence it is considered appropriate to derive no PNECs.

However, a confined environmental assessment was carried out for calculation of man via environment to ensure that the risk of exposure via the food-chain is controlled as well.

The results of exposure and risk assessments are covering all relevant releases throughout the life cycle. Comparison of all the derived DNELs with the results of the exposure assessment shows that exposures in all life cycle stages of the substance are always well below the derived DNELs and thus RCR values are always well below 1. As well for consumer use and indirect exposure of humans via the environment RCR values are always well below 1.

The final conclusion is based on the risk characterisation ratios (RCR) and on the qualitative assessment.

 

Robustness of Exposure and Risk Assessment:

The exposure and risk assessment for consumers and industrial and professional workers was carried out by use of the ECETOC TRA v.3.0 as being integrated in the ECHA Tool Chesar 2.3. The operational conditions and estimated exposures for workers were further compared with the COSHH benchmark concept to ensure adequate protection for workers. Consumer Exposure calculation was carried out by consideration of realistic worst case concentrations and worst case amounts of products used.

Due to the above procedure, the results and conclusions of the exposure and risk assessment are considered to be sufficiently robust for the purpose of this exposure based waiving.

 

In conclusion, further testing of intrinsic properties of Guanidine nitrate is not justified, especially when taking into account animal welfare reasons. In particular, as pursuant to Article 13 (1) of REACH Regulation, testing on vertebrate animals for the purpose of REACH in accordance with Annex VIII, Sections 8.6 and 8.7, Annex IX and Annex X may be omitted where justified by information on exposure and implemented risk management measures as specified in Annex XI, section 3.

Short description of key information:
A No Observed Adverse Effect Level (NOAEL) of 300 mg/kg body weight/day for fertility parameters was established from an oral sub-chronic toxicity study according to OECD guideline 408 with the read-across substance Guanidine hydrochloride.

Justification for selection of Effect on fertility via oral route:
Read-across data from a GLP compliant guideline study with reliability 1.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

90 day repeatded dose, sperm, oestrus cycle

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-03-27 to 2015-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents), adopted 21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 7-8 weeks old, males: 7-8 weeks old
- Weight at study initiation: females: 137 -170 g; males: 147 -193 g
- Housing: The animals were housed in groups (5 animals/sex/cage) in type IV cages
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1526) ad libitum
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: males From: 2014-03-25 to 2014-08-05 females From: 2014-03-25 to 2014-08-04

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-aqua ad injectionem (AlleMan Pharma)
- Concentration in vehicle: low dose: 5 mg/L; medium dose 20 mg/mL; high dose: 60 mg/mL
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples of at least 5 mL were retained from all groups in weeks 1, 5, 9 and 13
during the treatment period and stored between -15 and -35 °C. In total 16 samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period.
From the low, medium and high dose group, samples of dosing formulations were frozen after 0 hours and after 10 days (at room temperature) after the preparation and stored at -15 to -35 °C until analysis. In total 6 samples.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days per week

Details on study schedule:

Fertility parameters from a subchronic repeated dose toxicity study

Dose / conc.:

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

Remarks:

low dose

Dose / conc.:

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

Remarks:

intermediate dose

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

control: 15 animals per sex
high dose: 15 animals per sex
low dose: 10 animals per sex
mid dose: 10 animals per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: according to the results of a previous dose range finding study
- Rationale for animal assignment (if not random): Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partners.
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 28 days (females) or 29 days (males) following the last administration.

Positive control:

no

Parental animals: Observations and examinations:

Refer to entry under IUCLID section 7.5.1

Oestrous cyclicity (parental animals):

Daily over a period of 8 days, the estrous cycle of all female animals was examined 4, 8 and 12 weeks after the first administration.
In the recovery animals the estrous cycle was examined during the last week of the recovery period.

Sperm parameters (parental animals):

At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser
(TOX IVOS Version 13.0).
Therefore sperm from left vas deferens was transferred to 0.1% bovine serum albumin solution. For staining two drops of 1% aqueous Eosin-Y solution were mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying the slides were dipped into 0.1% acetic acid for approximately 30 seconds to intensify the colouring.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally, mainly as irregularly long cycles, in all treatment groups including control and irrespective of the duration of the treatment. This was considered incidental and not related to the treatment with the test item.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups neither in the percentage of motile, static or rapidly moving epididymal sperms nor in testicular number of sperms/g testis.
Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item.

For other parameters refer to IUCLID section 7.5.1 Robust Study Summary for repeated dose toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Conclusions:

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.

Executive summary:

In a subchronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), Guanidine hydrochloride was administered to 10 Wistar rats/sex/dose in water, by gavage at dose levels of 0, 50, 100 and 300 mg/kg bw/day.

In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects a satellite group of 5 rats/sex was exposed at dose levels of 0 and 300 mg/kg bw/day (control and HD).

To evaluate possible toxic effects on fertility, the estrous cycle was examined at defined time points of the treatment and recovery period and epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were evaluated at the end of the treatment and recovery period. Moreover, a detailed histopathological evaluation of the reproductive organs was performed.

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.

Reference 2

Endpoint:

fertility, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Read-across from a GLP guideline study with Reliability 1
Justification for read-across: Guanidine hydrochloride and guanidine nitrate dissociate in aqueous media to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the effects of the ions separately. A detailed justification is outlined in IUCLID chapter 13.2 "Justification for read-across".

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Limit test:

no

Analytical verification of doses or concentrations:

yes

Details on study schedule:

Fertility parameters from a subchronic repeated dose toxicity study

Positive control:

no

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Guanidin nitrate had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally, mainly as irregularly long cycles, in all treatment groups including control and irrespective of the duration of the treatment. This was considered incidental and not related to the treatment with the test item.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups neither in the percentage of motile, static or rapidly moving epididymal sperms nor in testicular number of sperms/g testis.
Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item.

For other parameters refer to IUCLID section 7.5.1 Robust Study Summary for repeated dose toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Conclusions:

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.
Based on read across it is assumed that also Gunaidiniumnitrate has no effect.

Executive summary:

In a subchronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), Guanidine hydrochloride was administered to 10 Wistar rats/sex/dose in water, by gavage at dose levels of 0, 50, 100 and 300 mg/kg bw/day.

In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects a satellite group of 5 rats/sex was exposed at dose levels of 0 and 300 mg/kg bw/day (control and HD).

To evaluate possible toxic effects on fertility, the estrous cycle was examined at defined time points of the treatment and recovery period and epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were evaluated at the end of the treatment and recovery period. Moreover, a detailed histopathological evaluation of the reproductive organs was performed.

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study was conducted according to modern regulatory standards and was adequately reported.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A No Observed Adverse Effect Level (NOAEL) of 350 mg/kg body weight/day for developmental toxicity was

established from a developmental toxicity study according to OECD guideline 414 with the read-across substance Guanidine hydrochloride.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-22 to 2014-10-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January, 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 11-12 weeks old (arrival), males: 19-20 weeks old (start of pairing)
- Weight at study initiation: females: 201 - 238 g; males: 330 - 449 g (start of pairing)
- Housing: The animals were kept individually in IVC cages
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1526) ad libitum
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: males From: 2014-02-13 to 2014-10-14 females From: 2014-04-15 to 2014-10-14

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-aqua ad injectionem (AlleMan Pharma)
- Concentration in vehicle: low dose: 10 mg/L; medium dose 30 mg/mL; high dose: 70 mg/mL
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first week of the study from the control, LD, MD and HD group (450 mg/kg as well as 350 mg/kg). Samples for analysis of the dose formulations of the test item were also taken in the last week of the study from the control, LD, MD and HD group (350 mg/kg).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day
- M/F ratio per cage: 1:2
The subsequent morning and each morning thereafter, the vaginal smear of each female was checked until positive evidence of mating was confirmed. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’.

Duration of treatment / exposure:

between gestation day 5 until gestation day 19

Frequency of treatment:

7 days per week

Duration of test:

On gestation day 20, sperm positive females were subjected to a caesarean section

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low Dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Medium Dose

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

High Dose

No. of animals per sex per dose:

control: 21 females
low dose (LD): 23 females
medium dose (MD): 23 females
high dose (HD): 24 females (8 females treated with 450 mg/kg BW/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
According to the results of the dose range finding study and in consultation with the sponsor the following doses were selected for the 3 dose groups:
LD = low dose: 50 mg/kg bw/d
MD = medium dose: 150 mg/kg bw/d
HD = high dose: 350 mg/kg bw/d
C= control group: vehicle
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

8 females from the HD group were treated with 450 mg/kg body weight/day from the start of the study for several treatment days (until 09 May 2014):
- animal no. 76 from GD 5 to GD 14 (euthanized on GD 14)
- animal no. 77 from GD 5 to GD 17 (found dead on GD 18)
- animal no. 78 from GD 5 to GD 14
- animal no. 79 from GD 5 to GD 10
- animal no. 80 from GD 5 to GD 9
- animal no. 81 from GD 5 to GD 9
- animal no. 82 from GD 5 to GD 8
- animal no. 83 from GD 5 to GD 7
After marked reduction of the body weight and food intake in those presumed pregnant animals of the HD group, the dose of the HD group was reduced to 350 mg/kg body weight/day. Females no. 84-99 of the HD group were treated with a dose of 350 mg/kg body weight/day for the whole treatment period.

- Rationale for animal assignment (if not random):

Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily all animals were observed
- Cage side observations: for morbidity and mortality except on weekends and public holidays when
observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, but no feeding study
-Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (gavage study)
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination or death during the study, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.

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 early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Historical control data:

Laboratory historical control data for prenatal development toxicity studies - wistar rat (year 2010 - 2014) reported within study report

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One female (no. 76) was euthanized in a moribund condition on gestation day 14 and another female of the HD group (no. 77) was found dead on gestation day 18. The animal of the HD group that was euthanized for welfare reasons on gestation day 14, showed marked loss of body weight, piloerection, moving the bedding and red nasal discharge. The animal of the HD group which was found dead on gestation day 18 lost body weight and was observed with slight to severe piloerection and a wasp waist. Females no. 76 and 77 were administered 450 mg/kg bw/d throughout their test participation.

The clinical sign of slight to severe piloerection was noted transiently in several females of the HD group treated with 450 to 350 mg/kg bw/d whereas only one female of the control group showed piloerection on one single day. Regarding females of the HD group which received 350 mg/kg bw/d throughout the whole treatment period, one female showed slight piloerection on one single day and another female was noted on two single days with slight or moderate piloerection. Thus, these findings in females treated with 450 to 350 mg/kg bw/d can be considered of minor toxicological relevance and in the remaining females as incidental. Slight to severe salivation was noted transiently with a dose-dependent pattern in most females of the HD group (at 450 to 350 mg/kg bw/d as well as at solely 350 mg/kg bw/d) and few females of the MD group. Moving the bedding was observed transiently in all females of the HD group, 10 females of the MD group and one female of the LD group. As the symptoms of salivation and moving the bedding were noted mainly immediately after dose administration, these signs were considered to be indicative of discomfort due to a local reaction to the test item. None of the females showed signs of abortion prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the LD and the MD group. However, mean body weight of the HD group was slightly lower compared to the control group from gestation day 8 onwards but without achieving statistical significance. Body weight gain was also slightly lower in the HD group compared to the control group on various intervals within the study period and achieved statistical significance on day 5-8 (p<0.001). The same effect was seen with statistical significance (p<0.01) when considering only those females of the HD group which received a dose of 350 mg/kg bw/d for the whole treatment period. This effect on the body weight and body weight gain of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be slightly lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from day 0 to day 20 (p<0.001) as well as on several treatment intervals. Food consumption was comparably reduced when considering only those females of the HD group with a continuous dosage of 350 mg/kg bw/d. Likewise, statistical significance was noted over the study period from day 0 to day 20 (p<0.01) as well as on several treatment intervals. This effect on the food consumption of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

Key result

Dose descriptor:

LOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Key result

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no test item-related effects of toxicological relevance noted on mean foetus weight, total number of foetuses, number of male and female foetuses, mean total litter weight and male and female litter weight.

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups.

A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls and litter incidences were statistically insignificant except for liver haematoma. Liver haematoma was noted with a statistically significantly higher litter incidence in the control group compared to the LD group without dose-dependency (C: 22%, LD: 0%, MD: 14%, HD: 10%). This was considered as incidental and not related to the treatment with the test item. As the remaining observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Craniofacial examination by a razor blade serial sectioning technique revealed few findings at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature.

Skeletal examination of the Alizarin red stained foetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparably lower in treated groups when compared to the control group. Statistically significant decrease in litter incidence of incomplete ossification of frontal bone (B) in HD and of 4th sternebra in HD, 14th full rib (B) in LD and supernumerary rib cartilage (B) in LD was observed when compared to the control group. Lack of dose dependency and consistency in these findings indicated no relevance with treatment. There was no indication of a compound-related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Prenatal parameters like group mean gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, number of live and dead foetuses, number of early and late resorptions, number of male and female foetuses, sex ratio and pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. Pregnancy rates were comparable between the groups and within the normal range of variation for animals of this strain and age.

Conclusions:

Under the condition of the study, 150 mg/kg body weight/day was considered as no observed adverse effect level (NOAEL) for maternal toxicity and 350 mg/kg body weight/day for developmental toxicity of Guanidine hydrochloride.

Executive summary:

In a developmental toxicity study according to OECD guideline 414 (adopted 22 January, 2001), Guanidine hydrochloride was administered to female Wistar rats during gestation days 5 to 19.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/day (21 females)

Low Dose: 50 mg/kg body weight/day (23 females)

Medium dose (MD) 150 mg/kg body weight/day (23 females)

High dose (HD): 350 mg/kg body weight/day (24 females)

Females were paired (ratio 1:2) for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

At a dose level of 450 mg/kg bw/d mortality was observed in two animals and clinical symptoms occurred in more animals than in the control group resulting in a reduction of the highest dosage to 350 mg/kg bw/d. Animals treated with 450 to 350 mg/kg bw/d as well as animals receiving 350 mg/kg bw/d throughout the whole treatment period were noted with a statistically significantly reduced body weight gain and food consumption.

No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level.

Maternal toxicity: NOAEL: 150 mg/kg bw/day

Based on mortality at 450 mg/kg bw/day; statistically significantly reduced body weight gain and food consumption (450 and 350 mg/kg bw/day)

Developmental toxicity: NOAEL: 350 mg/kg bw/day

No effects on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from a GLP guideline study with Reliability 1 Justification for read-across: Guanidine hydrochloride and guanidine nitrate dissociate in aqueous media to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the effects of the ions separately. A detailed justification is outlined in IUCLID chapter 13.2

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One female (no. 76) was euthanized in a moribund condition on gestation day 14 and another female of the HD group (no. 77) was found dead on gestation day 18. The animal of the HD group that was euthanized for welfare reasons on gestation day 14, showed marked loss of body weight, piloerection, moving the bedding and red nasal discharge. The animal of the HD group which was found dead on gestation day 18 lost body weight and was observed with slight to severe piloerection and a wasp waist. Females no. 76 and 77 were administered 450 mg/kg bw/d throughout their test participation.

The clinical sign of slight to severe piloerection was noted transiently in several females of the HD group treated with 450 to 350 mg/kg bw/d whereas only one female of the control group showed piloerection on one single day. Regarding females of the HD group which received 350 mg/kg bw/d throughout the whole treatment period, one female showed slight piloerection on one single day and another female was noted on two single days with slight or moderate piloerection. Thus, these findings in females treated with 450 to 350 mg/kg bw/d can be considered of minor toxicological relevance and in the remaining females as incidental. Slight to severe salivation was noted transiently with a dose-dependent pattern in most females of the HD group (at 450 to 350 mg/kg bw/d as well as at solely 350 mg/kg bw/d) and few females of the MD group. Moving the bedding was observed transiently in all females of the HD group, 10 females of the MD group and one female of the LD group. As the symptoms of salivation and moving the bedding were noted mainly immediately after dose administration, these signs were considered to be indicative of discomfort due to a local reaction to the test item. None of the females showed signs of abortion prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the LD and the MD group. However, mean body weight of the HD group was slightly lower compared to the control group from gestation day 8 onwards but without achieving statistical significance. Body weight gain was also slightly lower in the HD group compared to the control group on various intervals within the study period and achieved statistical significance on day 5-8 (p<0.001). The same effect was seen with statistical significance (p<0.01) when considering only those females of the HD group which received a dose of 350 mg/kg bw/d for the whole treatment period. This effect on the body weight and body weight gain of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be slightly lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from day 0 to day 20 (p<0.001) as well as on several treatment intervals. Food consumption was comparably reduced when considering only those females of the HD group with a continuous dosage of 350 mg/kg bw/d. Likewise, statistical significance was noted over the study period from day 0 to day 20 (p<0.01) as well as on several treatment intervals. This effect on the food consumption of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

Key result

Dose descriptor:

LOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Key result

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEC

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no test item-related effects of toxicological relevance noted on mean foetus weight, total number of foetuses, number of male and female foetuses, mean total litter weight and male and female litter weight.

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups.

A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls and litter incidences were statistically insignificant except for liver haematoma. Liver haematoma was noted with a statistically significantly higher litter incidence in the control group compared to the LD group without dose-dependency (C: 22%, LD: 0%, MD: 14%, HD: 10%). This was considered as incidental and not related to the treatment with the test item. As the remaining observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Craniofacial examination by a razor blade serial sectioning technique revealed few findings at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature.

Skeletal examination of the Alizarin red stained foetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparably lower in treated groups when compared to the control group. Statistically significant decrease in litter incidence of incomplete ossification of frontal bone (B) in HD and of 4th sternebra in HD, 14th full rib (B) in LD and supernumerary rib cartilage (B) in LD was observed when compared to the control group. Lack of dose dependency and consistency in these findings indicated no relevance with treatment. There was no indication of a compound-related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Prenatal parameters like group mean gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, number of live and dead foetuses, number of early and late resorptions, number of male and female foetuses, sex ratio and pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. Pregnancy rates were comparable between the groups and within the normal range of variation for animals of this strain and age.

Conclusions:

Under the condition of the study, 150 mg/kg body weight/day was considered as no observed adverse effect level (NOAEL) for maternal toxicity and 350 mg/kg body weight/day for developmental toxicity of Guanidine nitrate.

Executive summary:

In a developmental toxicity study according to OECD guideline 414 (adopted 22 January, 2001), Guanidine hydrochloride was administered to female Wistar rats during gestation days 5 to 19.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/day (21 females)

Low Dose: 50 mg/kg body weight/day (23 females)

Medium dose (MD) 150 mg/kg body weight/day (23 females)

High dose (HD): 350 mg/kg body weight/day (24 females)

Females were paired (ratio 1:2) for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

At a dose level of 450 mg/kg bw/d mortality was observed in two animals and clinical symptoms occurred in more animals than in the control group resulting in a reduction of the highest dosage to 350 mg/kg bw/d. Animals treated with 450 to 350 mg/kg bw/d as well as animals receiving 350 mg/kg bw/d throughout the whole treatment period were noted with a statistically significantly reduced body weight gain and food consumption.

No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level.

Maternal toxicity: NOAEL: 150 mg/kg bw/day

Based on mortality at 450 mg/kg bw/day; statistically significantly reduced body weight gain and food consumption (450 and 350 mg/kg bw/day)

Developmental toxicity: NOAEL: 350 mg/kg bw/day

No effects on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted according to modern regulatory standards and was adequately reported.

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

In a developmental toxicity study according to OECD guideline 414 (adopted 22 January, 2001), the read-across substance Guanidine hydrochloride was administered to female Wistar rats during gestation days 5 to 19 by gavage.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/day (21 females)

Low Dose: 50 mg/kg body weight/day (23 females)

Medium dose (MD) 150 mg/kg body weight/day (23 females)

High dose (HD): 350 mg/kg body weight/day (24 females)

Females were paired (ratio 1:2) for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

The first batches received 450 mg/kg bw/d at this dose level mortality was observed in two animals and clinical symptoms occurred in more animals than in the control group resulting in a reduction of the highest dosage to 350 mg/kg bw/d. Animals treated with 450 to 350 mg/kg bw/d as well as animals receiving 350 mg/kg bw/d throughout the whole treatment period were noted with a statistically significantly reduced body weight gain and food consumption.

No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level.

The NOAEL for maternal toxicity was 150 mg/kg bw/day, based on mortality at 450 mg/kg bw/day; statistically significantly reduced body weight gain and food consumption (450 and 350 mg/kg bw/day).

No effects on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level, the NOAEL for developmental toxicity was 350 mg/kg bw/day.

 

Justification for read-across:

Guanidine hydrochloride and guanidine nitrate dissociate in biological fluids to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the systemic intrinsic properties of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. Systemic effects of guanidine hydrochloride are expected to be based on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine hydrochloride for effects of the guanidine ion is considered valid. The effects of the nitrate ion will be discussed separately by read-across. This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies.

A more detailed justification is attached and outlined in CSR chapter 1.1.2 as well.

 

Effects of nitrate ions

Data are available from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 with Potassium nitrate. For the reproductive subgroup of the study 5 male and 10 female Sprangue-Dawley rats per dose were exposed at dose levels of 0, 250, 750 and 1500 mg/kg bw/day by gavage for a period of 28 (males) and approximately 53 (females) days.

There were no treatment-related deaths and no signs of overt clinical toxicity. There were no effects on body weight, food consumption, or food efficiency. Mating performance and fertility were unaffected by treatment. All animals mated within 4 days. There were no treatment-related effects on gestation length, gestation index, litter size, offspring survival indices, sex ration, offspring bodyweight, or macropathology for offspring.

The NOAEL for reproductive toxicity was established at the highest tested dose at 1,500 mg/kg bw/day (equivalent to 920 mg/kg bw/day nitrate)

 

Considering developmental toxicity the following is cited from the OECD SIAR, Nitrates Category, 2007:

“Potassium nitrate was given by gavage during gestation at doses up to 400 mg/kg (mice), up to 280 mg/kg (hamsters), up to 1980 mg/kg (rats) and up to 206 mg/kg (rabbits). No adverse effects of potassium nitrate were reported on nidation, maternal or fetal survival, or incidence of soft or skeletal tissue abnormalities. In a reproductive study in guinea pigs given potassium nitrate at concentrations of 300, 2,500, 10,000, and 30,000 ppm up to 204 days, the NOAEL for maternal reproductive toxicity was 10,000 ppm. In a two-generation rabbit study, sodium nitrate at dose levels of 0, 8, 250 or 500 mg/L in drinking water had no effect on the number of pregnancies, litter size or pup weights. Sodium nitrate was given by gavage during gestation at doses up to 400 mg/kg for groups of mice and hamsters and up to 250 mg/kg for groups of rats and rabbits. No effects of sodium nitrate were reported on nidation, maternal or fetal survival, or incidence of soft or skeletal tissue abnormalities.

Sodium nitrate did not induce abnormalities of sperm heads in mice treated for three days but following 14 days of treatment with sodium nitrate, sex chromosomal univalency and abnormal sperm-head frequency were significantly higher in mice. However, statistically significant reductions in fertility and litter size were not observed. Based on the available data, members of the nitrate category are not considered reproductive or developmental toxicants.”

 

In developmental toxicity studies for sodium nitrate and potassium nitrate conducted by FDA research laboratories (1972), in mice, rats, hamsters and rabbits no clearly discernible effect on nidation or on maternal or fetal survival were reported at the highest tested doses. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. The highest dose (400 mg/kg bw/d) was tested in mice for both substances and in hamsters for sodium nitrate. Studies in other animals were conducted with lower doses.

 

Conclusion

No effect on reproductive toxicity was observed in the Screening study with Potassium nitrate and a NOAEL was 1500 mg/kg bw/day (equivalent to 920 mg/kg bw/day nitrate), confirmed by OECD SIAR, Nitrates Category, 2007.

In the developmental toxicity study with Guanidine hydrochloride no effects on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level of 350 mg/kg bw/d.

As a dose level of 350 mg/kg bw/d of guanidine nitrate is equivalent to approx. 180 mg/kg bw/d nitrate, it is reasonable to conclude that effects revealed in the reproductive toxicity study at this dose level, are only caused by guanidinium toxicity.

Thus the derived NOAEL of 350 mg/kg bw/day is considered a reliable dose descriptor for developmental toxicity of guanidine nitrate. In particular, considering that adjustment of molecular weight will lead to higher value for guanidine nitrate.

For assessment of Guanidine nitrate the NOAEL of 100 mg/kg bw/day obtained for repeated dose toxicity from the sub-chronic toxicity study is considered the reliable dose descriptor, the respective DNELs are also protective for developmental toxicity.

 

Pre-natal developmental toxicity in a second species

The conduct of a Pre-natal developmental toxicity study in a second species is omitted according to Annex XI section 3.2 of REACH Regulation (EC) No 1907/2006 based on an exposure based adaptation.

The rules outline in section 3.2 (a) of Annex XI were used for exposure based adaptation:

An exposure assessment according to Annex I of REACH Regulation was conducted. This assessment is built on the use of exposure scenarios covering all relevant stages of the entire life cycle and respective exposure estimates, including a qualitative and quantitative risk characterisation.

All of the following conditions are fulfilled:

 

(i) the results of the exposure assessment covering all relevant exposures throughout the life cycle of the substance demonstrate the absence of or no significant exposure in all scenarios of the manufacture and all identified uses as referred to in Annex VI section 3.5

 

(ii) a DNEL or a PNEC can be derived from results of available test data for the substance concerned taking full account of the increased uncertainty resulting from the omission of the information requirement, and that DNEL or PNEC is relevant and appropriate both to the information requirement to be omitted and for risk assessment purposes

 

(iii) the comparison of the derived DNEL or PNEC with the results of the exposure assessment shows that exposures are always well below the derived DNEL or PNEC;

 

The full comprehensive exposure assessment is outlined in chapters 9 and 10 of the chemical safety report (CSR). The spectrum of identified applications is very small and specific. The identified applications are use as intermediate, use in fertilizers and explosives. The results of exposure and risk assessment covering all relevant exposures throughout the life cycle of the substance demonstrate a low exposure in all scenarios of manufacture, formulation, professional use, consumer use and indirect exposure of humans via the environment.

The very small spectrum of identified uses as referred to in Annex VI section 3.5 is documented in chapters 9 and 10 of CSR. Especially consumer use is limited to infrequent use in fertilizers at very low concentrations of 0.2 % w/w and at low volume. Same is true for "service live" of use in airbags,  where an exposure to the substance through the activated airbag is very rare, short time and only of very low concentration as the guanidine nitrate is consumed in the ignition.

 

Strictly conservative DNELs have been derived taking full account of the increased uncertainty resulting from the omission of the information requirement.

Results from the available test data include a pre-natal developmental toxicity study in rats and a sub-chronic toxicity study in rats. In addition to guideline requirements an assessment of fertility parameters was conducted within the sub-chronic toxicity study, especially to support the robustness of exposure based adaptation. No effects on estrous cycle, epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were observed.

These data are considered to be appropriate and reliable dose descriptors for DNEL derivation for omitting a prenatal developmental toxicity study in the second species and a two-generation reproductive toxicity study.

No substance-related adverse effects regarding reproductive/developmental endpoints were found in any of the tests conducted. Effects on general toxicity were observed in acute toxicity studies and in the sub-chronic toxicity study. As well in the developmental toxicity study maternal toxicity was observed. Guanidine Nitrate has no genotoxic properties as proven in the full genotoxic test battery.

The NOAELs used to derive the DNELs for fertility and developmental toxicity correspond to the maximum doses tested. The DNELs fertility have been derived from results of the sub-chronic repeated dose toxicity study, taking full account of the potential increased uncertainty resulting from the omission of the information requirement by applying an additional assessment factor of 2. This additional assessment factor has also been applied to DNELs derived for developmental toxicity.

The derived DNELs for fertility and developmental toxicity are all higher than the DNELs derived based on the NOAEL observed for systemic toxicity in the sub-chronic toxicity study.

Accordingly, systemic toxicity was considered the most sensitive Endpoint and thus the reliable dose descriptor for further assessment. The derived DNELs are protective for fertility and developmental toxicity including the potential increased uncertainty resulting from the omission of the information requirement and thus are relevant and appropriate for both the information requirement to be omitted and for risk assessment purposes.

No classification according to GHS Regulation EC No 1272/2008 for acute or chronic aquatic toxicity is required for guanidine nitrate, hence it is considered appropriate to derive no PNECs.

However, a confined environmental assessment was carried out for calculation of man via environment to ensure that the risk of exposure via the food-chain is controlled as well.

The results of exposure and risk assessments are covering all relevant releases throughout the life cycle. Comparison of all the derived DNELs with the results of the exposure assessment shows that exposures in all life cycle stages of the substance are always well below the derived DNELs and thus RCR values are always well below 1. As well for consumer use and indirect exposure of humans via the environment RCR values are always well below 1.

The final conclusion is based on the risk characterisation ratios (RCR) and on the qualitative assessment.

 

Robustness of Exposure and Risk Assessment:

The exposure and risk assessment for consumers and industrial and professional workers was carried out by use of the ECETOC TRA v.3.0 as being integrated in the ECHA Tool Chesar 2.3. The operational conditions and estimated exposures for workers were further compared with the COSHH benchmark concept to ensure adequate protection for workers. Consumer Exposure calculation was carried out by consideration of realistic worst case concentrations and worst case amounts of products used.

Due to the above procedure, the results and conclusions of the exposure and risk assessment are considered to be sufficiently robust for the purpose of this exposure based waiving.

 

In conclusion, further testing of intrinsic properties of Guanidine nitrate is not justified, especially when taking into account animal welfare reasons. In particular, as pursuant to Article 13 (1) of REACH Regulation, testing on vertebrate animals for the purpose of REACH in accordance with Annex VIII, Sections 8.6 and 8.7, Annex IX and Annex X may be omitted where justified by information on exposure and implemented risk management measures as specified in Annex XI, section 3.

Justification for selection of Effect on developmental toxicity: via oral route:
Read-across data from a GLP compliant guideline study with reliability 1.

Justification for classification or non-classification

Reliable data from the read-across substance Guanidine hydrochloride are available for fertility and developmental toxicity. There is no evidence for toxicity to reproduction or developmental toxicity of Guanidine; further members of the nitrate category are not considered reproductive or developmental toxicants.

In conclusion guanidine nitrate does not need to be classified for reproductive toxicity according to CLO, EU GHS (Regulation (EC) No 1272/2008.